ES2273861T3 - IMMUNIZATION OF MILK VACCINE LIVESTOCK WITH GAPC PROTEIN AGAINST STREPTOCOCCUS INFECTIONS. - Google Patents

Abstract

The use of a GapC protein in the manufacture of the composition of a vaccine useful in the treatment or prevention of mastitis in a mammalian subject, in which the GapC protein is selected from the group consisting of: (a) a GapC protein isolated from Sreptococcus dysgalactiae consisting of the amino acid sequence shown at amino acid positions 1 to 336, both inclusive, of Figures 1A-1B (SEQ ID NO: No. 4) or an amino acid sequence with a sequence identity of at least 80% with the previous one; (b) a GapC protein isolated from Sreptococcus agalactiae consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 2A-2B (SEQ ID NO: 6) or an amino acid sequence with a sequence identity of at least 80% with the previous one; (c) a GapC protein isolated from Sreptococcus uberis consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 3A-3B (SEQ ID NO: 8) or a amino acid sequence with a sequence identity of at least 80% with the previous one; (d) a GapC protein isolated from Sreptococcus parauberant consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 4A-4B (SEQ ID NO: 10) or an amino acid sequence with a sequence identity of at least 80% with the previous one; (e) a GapC protein isolated from Sreptococcus iniae consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 5A-5B (SEQ ID NO: 12) or a amino acid sequence with a sequence identity of at least 80% with the previous one.

Description

Immunization of dairy cattle with GapC protein against Streptococcus infections.

Technical scope

The present invention relates, in general terms, to bacterial antigens and to the genes encoding them. More specifically, the present invention relates to the cloning, expression and characterization of the plasmin-binding GapC proteins from Streptococcus dysgalactiae, Streptococcus agalactiae, Streptococcus uberis, Streptococcus parauberis and Streptococcus initie , and their use in the composition composition vaccines

Background

Mastitis is an infection of the gland Breast usually caused by bacteria and fungi. The answer inflammatory that occurs after infection causes a decrease in yield and production quality of milk and causes considerable economic losses annually to the dairy industry.

Among the bacterial species most commonly associated with mastitis 20 species of the genus Streptococcus, including Streptococcus aureus, Streptococcus uberis (without typing), Streptococcus agalactiae (Lancefield group B), Streptococcus dysgalactiae (Lancefield group C), Streptococcus zooepidemicus are , and streptococci of the Lancefield D, G, L and N groups. Some of these species are infectious diseases (eg S. agalactiae ), while others are considered environmental pathogens (eg S. dysgalactiae and S. uberis ).

The environmental pathogen S. uberis is responsible for about 20% of all clinical cases of mastitis (Bramley, AJ and Dodd, FH [1984] J. Dairy Res. 51 : 481-512; Bramley, AJ [1987] Animal Health Nutrition 42 : 12-16; Watts, JL [1988] J. Dairy Sci . 71 : 1616-1624); it is the microorganism predominantly isolated from the mammary glands during non-lactating periods (Bramley, AJ [1984] Br. Vet. J. 140 : 328-335; Bramley and Dodd [1984] J. Dairy Res. 51 : 481-512; Oliver, SP [1988] Am. Vet. Res. 49 : 1789-1793).

Normally, the mastitis that occurs as a result of an infection with S. uberis is subclinical and is characterized by the production of a seemingly normal milk but which presents an increase in the somatic cell count due to the influx of leukocytes. The chemical composition of milk changes due to the suppression of secretion, with a transfer of sodium chloride and bicarbonate from the blood to the milk that causes a change towards a more alkaline pH. S. uberis mastitis also takes the form of an acute clinical disorder with obvious signs of disease such as the presence of clots and change of color in the milk and inflammation and hardening of the mammary gland. Some cases of this disease with clinical manifestation can be serious and pyrexia may occur. For a review of the clinical manifestations of mastitis by S. uberis, see: Bramley (1991) Mastitis: physiology or pathology , pp. 3-9, in C. Burvenich, G. Vandeputte-van Messom and AW Hill (ed.), New insights into the pathogenesis of mastitis , Rijksuniversiteit Gent, Belgium; and Schalm et al. (1971) The mastitis complex - A brief summary, pp. 1-3, in Bovine Mastitis , Lea & Febiger, Philadelphia.

Conventional control methods antibacterial such as disinfection of post-milking nipples and the Antibiotic treatment are effective in controlling many types of contagious mastitis, but the microorganisms present in the environment usually found in the stables of the cattle are usually resistant to these measures. Therefore, the Vaccination is an interesting strategy that prevents mammary gland infections and has proven to be beneficial in the case of some contagious pathogens that cause  mastitis.

There is limited literature in reference to vaccination studies with S. dysgalactiae and S. uberis and variable results have been observed. In some cases, immunization has resulted in increased vulnerability to the specific microorganism and, in other cases, protection against a specific strain has been obtained.

For example, in previous studies it has been shown that primary infection with S. uberis can significantly reduce the infection rate following a second challenge test with the same strain (Hill, AW [1988] Res. Vet. Sci. 44 : 386 -387). Local vaccination with dead microorganisms of S. uberis protects the mammary gland against infection with the homologous strain (Finch et al. (1994) Infect. Immun. 62 : 3599-3603). Similarly, it has been shown that subcutaneous vaccination with live microorganisms of S. uberis causes a dramatic modification of the pathogenesis of mastitis caused by the same strain (Hill et al. [1994] FEMS Immunol. Med. Microbiol. 8 : 109-118). Animals vaccinated in this way produce milk with a smaller amount of bacteria and many animals remain free of infection.

However, vaccination with live or attenuated strains may pose a risk to the recipient animal. In addition, it is clear that conventional vaccines of dead microorganisms are, in general, not at all effective against S. uberis and S. agalactiae , or because of their lack of protective antigens in cells in vitro or by masking said antigens by molecular mimicry .

The current lack of vaccines for mastitis caused by S. agalactiae and infectious streptococcal strains is due, at least in part, to a lack of knowledge regarding virulence determinants and protective antigens produced by those microorganisms that are involved in the invasion and protection of the mammary gland (Collins et al. [1988] J. Dairy Res. 55 : 25-32; Leigh et al. [1990] Res. Vet. Sci. 49 : 85-87; Marshall et al. ( 1986) J. Dairy Res. 53 : 507-514).

It is known that S. dysgalactiae binds to several plasma proteins such as fibronectin, fibrinogen, collagen, alpha-II-macroglobulin, IgG, albumin and other compounds. The microorganism also produces hyaluronidase and fibrinolysin and is able to adhere to the epithelial cells of the mammary glands and invade them. However, the exact roles played by these bacterial components responsible for these phenotypes in the pathogenesis are unknown.

Similarly, very little is known about the pathogenesis of S. uberis infection. In addition, the influence of virulence factors of S. uberis on defense mechanisms and the physiology of the mammary gland is not well defined. Among the virulence factors associated with S. uberis are the hyaluronic acid capsule (Hill, AW [1988] Res. Vet. Sci. 45 : 400-404), hyaluronidase (Schaufuss et al. [1989] Zentralbl. Bakteriol Ser. A 271 : 46-53), the R-type protein (Groschup, MH and Timoney, JF [1993] Res. Vet. Sci. 54 : 124-126) and a cohemolysin, the CAMP factor, also known as UBERIS factor (Skalka, B. and Smola, J. (1981) Zentralbl. Bakteriol. Ser. A 249 : 190-194), R-type protein, plasminogen activator and CAMP factor. However, very little is known of its functions in pathogenicity.

The use of Streptococcus virulence determinants as immunogenic agents has been proposed. For example, it has been shown that the S. uberis CAMP factor protects vertebrate organisms against infection by this microorganism (Jiang, US Patent No. 5,863,553).

The γ antigen of strain A909 (No. ATCC 27591) Group B streptococcus is a component of the complex protein C marker, which additionally consists of a α subunit and a β (Boyle, US Pat. no. 5 721 339). It has been described that subpopulations of serotypes Ia, II and practically all cell types of subtype Ib of group B stroptococci express protein components C. The use of γ subunits has been proposed as immunogenic agents against streptococcal infections from group B of Lancefield. However, its use in prevention and treatment of infections in animals, between they mastitis in cattle.

The M protein of group A streptococci is considered one of the main virulence factors of this microorganism due to its ability to prevent the attack of human phagocytes (Lancefield, RC [1962] J. Immunol. 89 : 307- 313). Bacteria persist in infected tissue until antibodies against the M molecule are produced. Antibodies specific to the M protein are capable of reversing the antifagocytic effect of the molecule and allow the elimination of the invading microorganism.

M proteins are one of the key virulence factors of Streptococcus pyogenes , due to the role they play in intervening in phagocytosis resistance (Kehoe, MA [1991] Vaccine 9 : 797-806) and their ability to induce harmful immune responses of host through its superantigenicity and its ability to induce cross-reactions between antibodies (Bisno, AL [1991] New Engl. J. Med. 325 : 783-793; Froude et al. [1989] Curr. Top. Microbiol. Immunol. 145 : 5-26; Stollerman, GH [1991] Clin. Immunol. Immunopathol. 61 : 131-142).

However, there are obstacles in the use of intact M proteins as vaccines. The opsonic epitopes of the protein are extremely specific, so they provide a very limited specific protection. In addition, some M proteins contain epitopes that cause a cross-reaction with tissues of the immunized individual, leading to a harmful autoimmune response (See eg, Dale, JL and Beached, GH [1982] J. Exp. Med. 156 : 1165 -1176; Dale, JL and Beached, GH [1985] J. Exp. Med. 161 : 113-122; Baird, RW, Bronze, MS, Drabs, W., Hill, HR, Veasey, LG and Dale, JL [ 1991] J. Immun. 146 : 3132-3137; Bronze, MS and Dale, JL [1993] J. Immun. 151 : 2820-2328; Cunningham, MW and Russell, SM [1983] Infect. Immun. 42 : 531- 538).

Chimeric proteins containing three different fibronectin binding domains derived from the fibronectin binding proteins of S. dysgalactiae and Staphylococcus aureus have been expressed on the surface of Staph cells . carnosus In the case of one of those proteins, intranasal immunizations with live Staph cells . Recombinant carnosus expressing the chimeric protein on its surface resulted in an improvement in the antibody response against a model immunogen present in the surface chimeric protein.

Previously, a plasmin-binding GapC protein from a Streptococcus group A strain has been identified and characterized and its use has been described in thrombolytic treatments (Boyle, et al. , U.S. Patent No. 5,237,050; Boyle, et al. . , US Patent No. 5 328 996).

However, to date neither the protective capacity of GapC has been studied nor the GapC proteins of Streptococcus dysgalactiae, Streptococcus agalactiae, Streptococcus uberis, Streptococcus parauberis or Streptococcus iniae have been isolated or characterized.

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Summary of the Invention

Accordingly, the present invention provides a novel use of GapC protein in manufacturing of the composition of a vaccine useful for treatment and prevention of mastitis in mammals; in this process the protein GapC is selected from the following group:

(a) A GapC protein isolated from Streptococcus dysgalactiae consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 1A-1B (SEQ ID NO: 4) or a amino acid sequence with at least 80% similarity to it.

(b) A GapC protein isolated from Streptococcus agalactiae consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 2A-2B (SEQ ID NO: 6) or a amino acid sequence with at least 80% similarity to it.

(c) A GapC protein isolated from Streptococcus uberis consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 3A-3B (SEQ ID NO: 8) or a amino acid sequence with at least 80% similarity to it.

(d) A GapC protein isolated from Streptococcus parauberis consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 4A-4B (SEQ ID NO: 10) or a amino acid sequence with at least 80% similarity to it.

(e) A GapC protein isolated from Streptococcus iniae consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 5A-5B (SEQ ID NO: 12).

In another embodiment, the invention relates to the use of a polynucleotide consisting of a sequence of GapC protein for the manufacture of a vaccine composition useful for the treatment and prevention of mastitis in mammals; in which the GapC protein is selected from the following group:

(a) A GapC protein isolated from Streptococcus dysgalactiae consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 1A-1B (SEQ ID NO: 4) or a amino acid sequence with at least 80% similarity to it.

(b) A GapC protein isolated from Streptococcus agalactiae consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 2A-2B (SEQ ID NO: 6) or a amino acid sequence with at least 80% similarity to it.

(c) A GapC protein isolated from Streptococcus uberis consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 3A-3B (SEQ ID NO: 8) or a amino acid sequence with at least 80% similarity to it.

(d) A GapC protein isolated from Streptococcus parauberis consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 4A-4B (SEQ ID NO: 10) or a amino acid sequence with at least 80% similarity to it.

(e) A GapC protein isolated from Streptococcus iniae consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 5A-5B (SEQ ID NO: 12) or a amino acid sequence with at least 80% similarity to it.

In addition the vaccine composition must contain an adjuvant.

Brief description of the figures

Figures 1A-1B show the isolated nucleotide sequences and deduced amino acid sequences of the gapC gene of S. dysgalactiae (SEQ ID NO : 3 and SEQ ID NO : 4).

Figures 2A-2B show the isolated nucleotide sequences and deduced amino acid sequences of the gapC gene of S. agalactiae (SEQ ID NO : 5 and SEQ ID NO : 6).

Figures 3A-3B show the isolated nucleotide sequences and deduced amino acid sequences of the S. uberis gapC gene (SEQ ID NO : 7 and SEQ ID NO : 8).

Figures 4A-4B show the isolated nucleotide sequences and deduced amino acid sequences of the gapC gene of S. parauberis (SEQ ID NO : 9 and SEQ ID NO : 10).

Figures 5A-5B show the isolated nucleotide sequences and deduced amino acid sequences of the gapC gene of S. iniae (SEQ ID NO : 11 and SEQ ID NO : 12).

Figures 6A-6E show a DNA alignment created by PileUp and displayed by Pretty software (a component of the GCG Wisconsin Package, version 10, supplied by the SeqWeb sequence analysis package, version 1.1, of the Canadian Bioinformatics Resource). The figure shows the nucleotide sequences isolated from the gapC genes of S. dysgalactiae (DysGapC, Check 9344), S. agalactiae (AgalGapC, Check 2895), S. uberis (UberGapC, Check 5966), S. parauberis (PUberGapC, Check 9672) and S. iniae (IniaeGapC, Check 990). Also included are the previously known sequences of S. equisimilis (SeqGapC, Check 5841), S. pyogenes (SpyGapC, Check 4037) and a bovine GAPDH protein (BovGapC, check 5059). The parameters of length and weight and are the same for all sequences (1018 and 1.00; respectively). The parameters used for the comparison of DNA sequences were the following: Plurality-2.00; Threshold-1; AveWeight-1.00; AveMatch-1.00; AvMisMatch-0.00; Symbol comparison table-pileupdna.cmp; CompCheck-6876; GapWeight-5; GapLengthWeight-1; PileUp MSF-10l8; Type-N; Check-3804 In the figure, the stripes represent identical nucleotides; the points represent gaps introduced by the software used to generate the alignment diagram; and the tildes (\ sim) represent regions not included in the total alignment due to differences in the length of the gene sequence.

Figures 7A-7B show an alignment of an amino acid sequence created by PileUp and shown by Pretty (same as the previous Figure) showing the amino acid sequences deduced from GapC proteins of S. dysgalactiae (DysGapC, Check 6731) , S. agalactiae (AgalGapC, Check 1229), S. uberis (UberGapC, Check 8229), S. parauberis (PUberGapC, Check 8889) and S. iniae (IniaeGapC, check 8785). Also known are the sequences of S. equisimilis (SeqGapC, Check 8252), S. pyogenes (SpyGapC, Check 6626) and a bovine GAPDH protein (BovGapC, Check 8479). In the figure, the stripes represent identical amino acid residues; the dots represent gaps introduced by the PileUp software; and the tildes (\ sim) represent regions not included in the total alignment due to differences in the length of the gene sequences.

Figure 8 shows the hydropathy diagrams on the Kyte-Doolittle scale (mean over a window of 7), Emini surface probability diagrams, Karplus-Schulz chain flexibility diagrams, Jameson antigenicity index diagrams -Wolf and diagrams Chou-Fasman the Garnier-Osguthorpe and-Robson secondary structure plots for the GapC protein isolated from S. dysgalactiae.

Figure 9 shows the hydropathy diagrams on the Kyte-Doolittle scale (average over a window of 7), Emini surface probability diagrams, Karplus-Schulz chain flexibility diagrams, Jameson antigenicity index diagrams -Wolf and diagrams of the secondary structure Chou-Fasman and Garnier-Osguthorpe-Robson for GapC protein isolated from S. agalactiae .

Figure 10 shows the hydropathy diagrams on the Kyte-Doolittle scale (average over a window of 7), Emini surface probability diagrams, Karplus-Schulz chain flexibility diagrams, Jameson antigenicity index diagrams -Wolf and diagrams of the secondary structure Chou-Fasman and Garnier-Osguthorpe-Robson for GapC protein isolated from S. uberis .

Figure 11 shows the hydropathy diagrams on the Kyte-Doolittle scale (average over a window of 7), Emini surface probability diagrams, Karplus-Schulz chain flexibility diagrams, Jameson antigenicity index diagrams -Wolf and diagrams of the secondary structure Chou-Fasman and Garnier-Osguthorpe-Robson for GapC protein isolated from S. parauberis .

Figure 12 shows the hydropathy diagrams on the Kyte-Doolittle scale (average over a window of 7), Emini surface probability diagrams, Karplus-Schulz chain flexibility diagrams, Jameson antigenicity index diagrams -Wolf and diagrams of the secondary structure Chou-Fasman and Garnier-Osguthorpe-Robson for GapC protein isolated from S. iniae .

Figure 13 is a graphical representation of the secondary structure Chou-Fasman diagrams for GapC protein isolated from S. dysgal .

Figure 14 is a graphical representation of the secondary structure Chou-Fasman diagrams for GapC protein isolated from S. agalactiae .

Figure 15 is a graphical representation of the secondary structure Chou-Fasman diagrams for GapC protein isolated from S. uberis .

Figure 16 is a graphical representation of the secondary structure Chou-Fasman diagrams for GapC protein isolated from S. parauberis .

Figure 17 is a graphical representation of the secondary structure Chou-Fasman diagrams for GapC protein isolated from S. iniae .

Figure 18 shows the results of an SDS-polyacrylamide gel electrophoresis of the recombinant S. dysgalactiae GapC protein produced in E. coli DE3. Lane 1, molecular weight marker (20.5-103 kDa; BioRad, Emeryville, CA); lane 2, soluble recombinant GapC protein of S. dysgalactiae purified by affinity chromatography in Ni-NTA. The numbers to the left of the figure indicate the positions of the molecular weight markers (in KDa).

Figure 19 is a histogram comparing the enzymatic activity of: bovine plasmin bound to intact S. dysgalactiae cells, bovine plasmin bound to recombinant S. dysgalactiae GapC protein purified by affinity chromatography and intact S. dysgalactiae cells , recombinant S. dysgalactiae GapC protein purified by affinity chromatography and bovine plasmin, all three separately. Enzymatic activity was measured by absorbance increases at 405 nm after the release of paranitroanilide from its synthetic substrate Chromozym-PL (Roche Diagnostics, Laval, Quebec, Canada). The results represent the average of three independent trials.

The changes in the percentage of quarters of udders infected by S. dysgalactiae over a period of seven days in three experimental groups are compared in Figure 20: (1) control animals without vaccinations; (2) animals vaccinated with Fc binding Mig protein; and (3) animals vaccinated with GapC. Infection was defined as the appearance of> 500 cfu of S. dysgalactiae per ml in secreted milk.

Figure 21 represents the maximum number of S. dysgalactiae in any quarter of udder versus titration of serum anti-GapC antibodies (expressed as the reciprocal at dilution showing activity above baseline levels). The anti-GapC titers were correlated with the maximum number of cfu recovered from the mammary glands r2 = 0.74, calculated with the GraphPadPrism computer application, v. 2.01, from GraphPad Software, Inc., San Diego, CA).

Figure 22 represents the cumulative number of affected mammary quarters against antibody titration (again expressed as the reciprocal of the dilution it shows activity above baseline levels). One was observed very good correlation between serum antibody levels anti-GapC and the total number of rooms infected.

Figure 23 illustrates the presence of bacteria in animals immunized with GapC; each point represents the number medium of bacteria present per ml of milk with respect to time (in days after provocation). In Figure 20, diamonds (- \ blacklozenge-) represent unvaccinated animals; the squares (- \ blacksquare-) represent animals with a title low (i.e. animals that show the worst response against to GapC based on its antibody titer), and the triangles (- \ Delta-) represent animals with a high titer (it is say, the rest of animals).

Figure 24 shows the presence of S. dysgalactiae in animals immunized with GapC, represented as the percentage of infected breast rooms versus time in days after provocation. In the figure, the dotted bars represent the unvaccinated animals; the bars with diagonal stripes represent the animals with low titers (that is, the animals that show the worst response against GapC based on their antibody titer); and the white bars represent the animals with high titles (that is, the rest of the animals).

Figure 25 shows the observed inflammatory responses caused by infection with S. dysgalactiae , represented as the mean somatic cell counts (RCS) for each experimental group versus time in days after provocation. In the figure, diamonds (- \ blacklozenge-) represent the rooms without vaccine or provocation; the squares (- \ blacksquare-) represent unvaccinated animals to which a provocation has been made; the triangles (- Δ-) represent the animals vaccinated with Mig and subjected to provocation; and the x (-X-) represent the animals vaccinated with GapC and subjected to provocation.

Figure 26 illustrates cell counts somatic per room on day 1 after provocation. In the figure, The bar represents the average for each group. The squares represent (- \ blacksquare-) unvaccinated animals; the triangles represent (- \ ding {115} -) animals vaccinated with GapC; and inverted triangles (- \ ding {116} -) animals vaccinated with Mig.

Figure 27 shows cell counts somatic animals with high levels not vaccinated, without provocation and immunized with GapC (i.e., the four animals who presented the highest antibody titers of the eight animals of each group) for a period of seven days after provocation, represented as the log_ {10} of the counts somatic cell media per ml of milk with respect to time in days after provocation. the diamonds (- \ blacklozenge-) they represent animals without vaccine or provocation; the squares (- \ blacksquare-) represent unvaccinated animals that a provocation has been made; the triangles (- \ Delta-) represent animals vaccinated with GapC and subjected to provocation.

Detailed description of the invention

The implementation of the present invention will use, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology and immunology, which are among the techniques of each field. These techniques are widely described in the literature. See, p. eg, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual , Vols. I, II and III, Second Edition (1989); Perbal, B., A Practical Guide to Molecular Cloning (1984); the series, Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); and Handbook of Experimental Immunology , Vols. I-IV (DM Weir and CC Blackwell eds., 1986, Blackwell Scientific Publications).

Throughout the text, the following abbreviations of amino acids:

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Alanina: Wing (TO) Arginine: Arg (R) Asparagine: Asn (N) Aspartic Acid: Asp (D) Cysteine: Cys (C) Glutamine: Gln (Q) Glutamic Acid: Glu (AND) Glycine: Gly (G) Histidine: His (H) Isoleucine: Ile (1) Leucine: Leu (L) Lysine: Lys (K) Methionine: Met (M) Phenylalanine: Phe (F) Prolina: Pro (P) Serina: Being (S) Threonine: Thr (T) Tryptophan: Trp (W) Tyrosine: Tyr (Y) Valine: Val (V)

1. Definitions

In the description of the present invention, they will use the following terms that are defined as indicated later.

It should be noted that, as used in this specification and in the appended claims, the singular forms "a / a" and "he / she" include plural referents unless clearly stated otherwise. Thus, for example, the reference to " Streptococcus GapC protein" includes a mixture formed by two or more of said proteins, and so on.

The terms "GapC protein" and "plasmin-binding GapC protein" (which are used interchangeably herein) or a nucleotide sequence encoding it, refer to a protein or nucleotide sequence, respectively, that derive of the GapC gene present in a variety of Streptococcus species, including, without limitation, certain strains of group A streptococci (Lottenbery, R., et al. [1987] Infect. Immun . 55 : 1914-1918). The figures show the nucleotide sequence of the Streptococcus representative gapC genes and the corresponding amino acid sequences of the GapC proteins that encode those genes. In particular, Figures 1 through 5 show the nucleotide sequences and amino acid sequences isolated from S. dysgalactiae (SEQ ID NO : 3 and SEQ ID NO : 4, respectively), S. agalactiae (SEC ID No.: 5 and SEC ID No.: 6, respectively), S. uberis (SEC ID No.: 7 and SEC ID No.: 8 , respectively), S. parauberis (SEQ ID No. 9: and SEQ ID No. 10, respectively) and S. iniae (SEQ ID No. 11 and SEC ID. No.: 12, respectively). However, a GapC protein as described herein is not limited to the sequences shown, since subtypes of each of those Streptococcus species are known and variations will exist between them.

Representative gapC genes, derived from S. dysgalactiae, S. agalactiae, S. uberis and S. parauberis , are found in plasmids pETl5bgapC (No. ATCC), pMF52lc (No. ATCC), pMF52la (No. ATCC), pMF52ld ( ATCC No.) and pMF52le (ATCC No.), respectively.

In addition, the derived protein or nucleotide sequences do not necessarily have to come from the gene described above, but can be produced by any other method such as, e.g. eg chemical synthesis, isolation (eg from S. dysgalactiae ) or by production in recombinant systems, based on the information presented in this document. In addition, the term is intended for proteins that possess amino acid sequences with substantial homology (as defined below) with respect to contiguous amino acid sequences encoded by genes that provide immunological or plasmin binding activity.

Thus, the terms are intended for both complete sequences such as partial sequences, truncated and immunogenic; as well as precursor forms and analogues protein active They are also included in the term nucleotide fragments of the gene that have at least about of eight adjacent base pairs; a minimum of more is preferred between 10 and 20; and, even more, a minimum of between 25 and 50 (or more) pairs of contiguous bases of the gene, or any integer between those values. Such fragments are useful as probes in the methods of diagnosis that will be discussed in detail later.

The terms also include those forms that have, as well as those that do not, a signal sequence if this it is present, in addition to the nucleic acid sequences that the encode In addition, the term is intended for protein forms of GapC that do not have an anchoring region to the membrane and to the nucleotide sequences encoding proteins with said deletions These deletions may be appropriate in systems that They do not allow protein secretion. In addition, the domains of protein plasmin binding may be present or not be Thus, for example, if the plasmin-binding GapC protein it is used to purify plasmin, generally the plasmin binding domain. If the protein is used in vaccine compositions, epitopes will be present immunogenic, which may include plasmin binding domains or not include them.

The terms also include proteins in their neutral form or in the form of acidic and basic addition salts Depending on the mode of preparation. Said acid addition salts they can be formed with amino groups and said basic salts with free carboxyls. Salts will be discussed in detail later Acid and basic pharmaceutically acceptable additions. Further, proteins can be modified by combining with others biological materials, such as lipids (both those that appear in shape natural with the molecule like other lipids that don't destroy the immune response) and polysaccharides; or by modification of the side chains, such as the acetylation of amino groups, the phosphorylation of hydroxyl side chains, oxidation groups  sulfhydryl, glycosylation of amino acid residues, as well as other modifications of the coded primary sequence.

The term, therefore, is intended to deletions, additions and substitutions in the sequence, provided and when the polypeptide is able to produce a response immune as defined in this document. In that sense, the substitutions that are especially preferred will be, in general, conservative in nature, that is, these substitutions will have place within a family of amino acids. For example, the Amino acids are generally divided into four families: (1) acids -glutamate and aspartate; (2) basic -lisin, arginine and histidine; (3) apolar-alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan; and (4) apolar without load -glycine, asparagine, glutamine, cystine, serine, threonine and tyrosine Phenylalanine, tryptophan and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonable to predict that isolated leucine replacement by isoleucine or valine, and vice versa; an aspartate by glutamate, and vice versa; a threonine for a serine, and vice versa; or a replacement similar conservative of one amino acid to another amino acid structurally related, do not imply an important effect on the biological activity Therefore, proteins that possess a amino acid sequence substantially similar to the molecule of reference, but that have unimportant substitutions of amino acids that do not substantially affect immunogenicity or protein affinity to bind plasmin, they are found within the definition of the reference polypeptide.

For example, the polypeptide of interest may include up to 5 to 10 conservative substitutions or non-conservative amino acids, or even up to a maximum of between 15 and 25 or 20 and 50 conservative substitutions or not conservative, or any integer between those values, always and when the function of the molecule remains intact.

In that sense, GapC proteins isolated from streptococci show several variable regions in their sequences of amino acids, located at positions 62 through 81; from 102 to 112; from 165 to 172; from 248 to 271; and of the 286 to 305.

These regions, which in S. dysgalactiae, S. agalactiae, S. uberis, S. parauberis and S. iniae show substitutions in amino acids 1 to 9, are susceptible to variation without substantially affecting the enzyme or immunogenic function.

Also, the substitutions that happen in the transmembrane binding domain, if present, and in the sequence signal, if present, normally does not affect the immunogenicity A person skilled in the art can determine easily other regions of the molecule of interest that can tolerate changes in reference to protein structure shown in figures 8 through 17 present in this document.

The term "streptococcal GapC protein" is intended for plasmin binding proteins, as defined above, which are derived from streptococcus species that produce it. Among them, although they are not limited to them, are the following species: S. dysgalactiae, S. agalactiae, S. uberis, S. parauberis and S. iniae . For example, a " S. dysgalactiae GapC protein" is a plasmin-binding GapC protein, as defined above, which is derived from S. dysgalactiae . Also, a "GapC protein of S. agalactiae " refers to a GapC binding protein that is derived from S. agalactiae .

The terms "wild" proteins or "native" refers to proteins or polypeptides isolated from the source in which proteins appear naturally. The "recombinant" polypeptides are those polypeptides produced by recombinant DNA techniques; that is to say, produced from cells transformed by a construct of Exogenous DNA encoding the desired polypeptide. Polypeptides "synthetics" are those that are prepared by synthesis chemistry.

The term "isolated" protein or polypeptide refers to a protein or polypeptide molecule that is separated and differentiated from the whole organism in which it find the molecule naturally; or a protein or polypeptide devoid, totally or partially, of sequences normally associated with it naturally; or a sequence in its natural form, but it has heterologous sequences (as defined below) in association with it.

The term "functionally equivalent" is refers to an amino acid sequence of a GapC protein of plasmin binding that triggers an immune response substantially equivalent or improved, as defined previously, compared to the response triggered by the plasmin binding GapC protein that is equal to GapC protein binding to reference plasmin or an immunogenic portion of the same.

The term "epitope" refers to the place of the antigen or hapten to which specific B or T lymphocytes respond. The term is also interchangeable for "determinant. antigenic "or" antigenic site. " antibodies that bind to the same epitope through an immunoassay that has the ability of an antibody to block the binding of another antibody by binding to a target antigen.

The term "immunogenic" protein or polypeptide refers to an amino acid sequence that triggers an immune response as described above. An "immunogenic" protein or polypeptide, as used herein, includes the complete sequence of the plasmid-binding GapC protein in question with any or none of the signal sequence, the membrane-binding domain, the domain. of plasmin binding and analogs or immunogenic fragments thereof. The term "immunogenic fragment" refers to a fragment of the plasmin-binding GapC protein that includes one or more epitopes and thus triggers the immune response described above. Such fragments can be identified using different techniques well known in the field for mapping epitopes. See, p. eg, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Monis, Ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes can be determined by, e.g. eg, the simultaneous synthesis of a large number of peptides on solid supports, peptides corresponding to portions of the protein molecule, and reacting said peptides with the antibodies while the former are still bound to the support. Such techniques are known in the field and are described in, e.g. eg, U.S. Patent No. 4 708 871; Geysen et al. (1984) Proc. Natl Acad. Sci. USA 81 : 3998-4002; Geysen et al. (1986) Molec. Immunol 23 : 709-715. Similarly, conformational epitopes are easily identified through the determination of the spatial conformation of amino acids by, e.g. eg, X-ray crystallography or two-dimensional nuclear magnetic resonance. See, p. eg, Epitope Mapping Protocols, supra . Antigenic regions of the proteins can also be identified using standard antigenicity and hydropathy diagrams, calculated with, e.g. eg, the Omiga version 1.0 program available at Oxford Molecular Group. This computer application uses the Hopp / Woods method (Hopp et al. , Proc. Natl. Acad. Sci USA [1981] 78 : 3824-3828) to determine antigenicity profiles, and the Kyte-Doolittle technique (Kyte et al , J. Mol. Biol. [1982] 157 : 105-132) for hydropathy diagrams. Figures 8 through 12 present in this document show the Kyte-Doolittle profiles of the representative proteins included in the invention.

For the purposes of the present invention, the immunogenic fragments will generally include at least three amino acids, preferably at least five amino acids, plus preferably an approximate minimum of between 10 and 15 amino acids, and, even more preferably, 25 or more amino acids of the molecule corresponding to the plasmatic binding GapC parental protein. Do not there is a critical upper limit on the length of the fragment, which can comprise close to the entire protein sequence or even be a fusion protein consisting of two or more epitopes of GapC

An "immunogenic composition" is a composition that consists of an antigenic molecule that when administers to a subject causes the development in said subject of a humoral or cellular immune response to the antigenic molecule of interest.

The term "composition with subunits of the vaccine "refers to a composition that contains at least one immunogenic polypeptide, but not all antigens, derived from an antigen or an analogue from the pathogen of interest. Said composition is substantially free of cells or intact particles of the pathogen, or of the lysate of said cells or particles Thus, a "composition with vaccine subunits"  is prepared from purified polypeptides at least partially (preferably, fully purified) of the pathogen or recombinant analogues thereof. A composition with vaccine subunits may contain the antigenic subunit or the antigens of interest substantially free of other antigens or pathogen polypeptides.

It is understood by "pharmaceutically acceptable "or" pharmacologically acceptable "a material that it is not biologically, or in any other way, unwanted; that is to say, the material must be administered to an individual in a formulation or composition that does not cause any unwanted biological effect and that does not interact in a harmful way with any of the components of the composition that contains it.

An "immune response" to a composition or vaccine refers to the development in the host of a cellular and antibody-mediated immune response to composition or vaccine of interest. Generally, an "answer immune "includes one or more of the following effects, although is not limited to them: production of antibodies, B lymphocytes, helper T lymphocytes, suppressor T lymphocytes and T lymphocytes cytotoxic or T γ lymphocytes; directed specifically to the antigen or antigens present in the composition of the vaccine of interest. Preferably, the host will trigger a protective or therapeutic immune response so that it improve the resistance of the mammary gland to a new infection or the clinical severity of the disease will be reduced. Bliss protection will be demonstrated both by the reduction and by the lack of the symptoms that normally appear in the infected host or by reducing the time needed for Recovery.

"Nucleic acid immunization" refers to the introduction into a host cell of a nucleic acid molecule that encodes one or more antigens selected for in vivo expression of an antigen, antigens, an epitope or epitopes. The nucleic acid molecule can be introduced directly into the subject by injection, inhalation; by oral, intranasal, mucosal or similar administration; or it can be introduced ex vivo , into cells that have been removed from the host. In the latter case, the transformed cells are reintroduced into the subject in which an immune response can be triggered against the antigen encoded in the nucleic acid molecule.

The term "treatment", as used In this document, it refers both to the prevention of infection or reinfection (prophylaxis), such as reduction or elimination of the symptoms of the disease of interest (treatment).

The term "mastitis" refers to the inflammation of the mammary glands of mammals, including cows, sheep, goats, sows, mares and the like, caused by the presence of S. uberis . The infection is manifested by the infiltration of phagocytic cells in the gland. Generally, four distinct clinical types of mastitis are recognized: (1) over-acute, associated with edema, heat, pain, abnormal secretion of the gland accompanied by fever and other signs of systemic alteration, such as noticeable depression, rapid and weak pulse, sunken eyes , weakness and complete anorexia; (2) acute, with changes in the gland similar to those described above but with fever, anorexia and depression between mild and moderate; (3) subacute, systemic changes do not occur and changes in the gland and its secretion are less marked; and (4) subclinical, the inflammatory reaction is detected only by standard tests for mastitis.

Standard tests for mastitis detection include the following, but are not limited to: the California mastitis test, the Wisconsin mastitis test, the Nagase test, the electronic cell count and the somatic cell count used to detect the persistence of a high leukocyte content in milk. In general, a somatic cell count of about 300,000 to 500,000 cells per ml, or higher, in milk is indicative of the presence of infection. Thus, a vaccine is considered effective in the treatment or prevention of mastitis when, e.g. For example, the somatic cell count in milk is kept below 500,000 cells per ml. For a discussion of mastitis and its diagnosis see, p. eg, The Merck Veterinary Manual: A Handbook of Diagnosis, Therapy, and Disease Prevention and Control for the Veterinarian , Merck and Co., Rahway, New Jersey, 1991.

The terms "vertebrate", "subject" or "vertebrate subject" refers to any member of the subfilum of the ropes and include, without limitation, the following: mammals, such as cows, sheep, pigs, goats, horses, humans and pets such as dogs and cats; birds, including domestic, wild and hunting, like roosters, chickens, ducks, turkeys and other chicken birds; and fish The term It does not denote any particular age. Thus, it is intended to cover both adult and newborn animals, as well as fetuses.

A molecule of "nucleic acids" can include, but is not limited to, the following: sequences prokaryotic, eukaryotic mRNA, cDNA from mRNA eukaryotic, eukaryotic DNA genomic sequences (e.g. from mammals) or even synthetic DNA sequences. The term it also includes sequences that consist of any of the DNA or RNA base analogs.

An "isolated" nucleic acid molecule is a nucleic acid molecule separated and differentiated from complete microorganism in which it is found naturally; or a nucleic acid totally or partially devoid of sequences normally associated with it naturally; or one sequence in the form that exists in nature, but which has heterologous sequences (as defined below) in association with her. The term "isolated" applied to polynucleotides refers to the polynucleotide being isolated from chromosome with which it is normally associated and isolated from the sequence Complete genomics in which it appears normally.

A "purified polynucleotide" refers to a polynucleotide of interest or a fragment thereof that is found essentially free, that is, it contains less than around 50%, preferably less than about 70% and more preferably less than about 90% of the protein at which naturally associates said polynucleotide. The techniques Purification of polynucleotides of interest are known widely in the field and include, for example, the lysis of the cell containing the polynucleotide with a chaotropic agent, and separation of the polynucleotide, or polynucleotides, and proteins by ion exchange chromatography, chromatography of affinity and sedimentation based on density.

A "coding sequence" or a "coding nucleotide sequence" of a particular protein is a nucleotide sequence that is transcribed and translated in vivo into a polypeptide when it is under the appropriate regulatory elements. The limits of the coding sequence are determined by a start codon at the 5 'amino terminal end and an end codon at the 3' carboxy terminal end. The coding sequences may, among others, be: prokaryotic sequences, cDNA from eukaryotic mRNA, genomic sequences of eukaryotic DNA (eg from mammals) or even synthetic DNA sequences. A transcription termination sequence is normally located at the 3 'end of the coding sequence. A "complementary" sequence is one in which each nitrogen base in a particular nucleotide position is complementary to the nitrogen base that appears in the same position of the reference sequence. To illustrate this, the complementary basis of adenosine is thymine and vice versa; Similarly, cytosine is complementary to guanine and vice versa. Therefore, the complementary sequence of the 5'-ATGCTGA-3 'reference sequence would be 3'-TACGACT-5'.

A "wild" or "native" sequence, as used herein, refers to polypeptide coding sequences that are essentially, as found in nature, for example, the sequences encoding the S. dysgalactiae GapC protein . shown in Figures 1A-1B (SEQ ID NO: 4).

The term "recombinant", used here to describe nucleic acid molecules, it refers to a polynucleotide of genomic origin, cDNA, semisynthetic or synthetic, which by its origin or its manipulation: (1) is not associated with the polynucleotide with which it is associated in nature or with a fragment thereof; and (2) binds to a polynucleotide other than which is associated naturally. The term "recombinant", referred to a protein or polypeptide, means a polypeptide produced by the expression of a recombinant polynucleotide. "Recombinant host cells", "host cells", "cells", "cell lines", "cell cultures" and  other terms of that type denoting cell lines of prokaryotic microorganisms or eukaryotic cells as entities Unicellular, used interchangeably and refers to cells that can be, or have been, used as recipients of recombinant vectors or other DNA transfers and include the progeny of the original cells that have been transfected. It is understood that the progeny of a single parental cell does not have why be completely identical in its morphology or content Genomic or total to the original parental cell due to mutations accidental or deliberate. This definition includes the progeny that is similar enough to the parental cell to show the relevant property, such as the presence of a nucleotide sequence encoding the desired peptide, and it found encompassed in the terms defined above.

The term "homology" refers to the percent identity between two polynucleotides or two domains of polypeptide. Two polypeptide sequences or two DNA sequences they are "substantially homologous" to each other when they show, at least about 80 to 85%, preferably at less about 90%, and more preferably, at least approximately, between 95 and 98% similarity in the sequence to along the defined length of the molecules. As used in this document, substantially homologous also refers to the sequences that show the complete identity of the sequence specified polypeptide or DNA.

In general, the term "identity" refers to the exact, amino acid to amino acid or nucleotide to nucleotide correspondence of two polypeptide or DNA sequences, respectively. The percentage of identity can be determined through the direct comparison of the sequence information of two molecules by aligning these sequences, counting the exact number of correspondences between the two aligned sequences, dividing by the length of the shortest sequence and multiplying the result by 100. Easily available computer programs can be used to assist in the analysis, such as ALIGN, Dayhoff, MO in Atlas of Protein Sequence and Structure MO Dayhoff Ed., 5 Suppl. 3 : 353-358, National Biomedical Research Foundation , Washington, DC, which adapts the local homology algorithm of Smith and Waterman (1981) Advances in Appl. Math 2 : 482-489 for peptide analysis. There are programs to determine the identity of the nucleotide sequences that are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, WI); for example, the BESTFIT, FASTA and GAP programs that are also based on the Smith and Waterman algorithm. These programs are easy to use with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package mentioned above. For example, the percent identity of a nucleotide sequence with respect to a reference sequence can be determined using the Smith and Waterman homology algorithm with a default score table and a penalty penalty of six nucleotide positions.

Another method for establishing percent identity in the context of the present invention is the use of the MPSRCH software package, whose rights to author belong to the University of Edinburgh which was developed by John F. Collins and Shane S. Sturrok and is distributed by IntelliGenetics, Inc. (Mountain View, CA). With these packages you can use the algorithm Smith-Waterman in which the default parameters are used for the score table (for example, penalty of 12 for opening of gap, penalty of one for extension of gap and a gap of six). From the data generated the value of "correspondence" reflects the "sequence identity". Other appropriate programs are known in the field for calculating the percent identity or similarity between sequences as, by example, BLAST, another alignment program that is used with default parameters. For example, BLASTN and BLASTP can be use using the following default parameters: code standard genetic; without filters; strands = both; cut point = 60; expected = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sorted by = highest score; Databases = not redundant; GenBank + EMBL + DDBJ + PDB + translations of GenSank + Swiss protein + Spupdate + PIR CDS. The details about These programs can be found at the following address Internet: http://www.ncbi.nlm.gov/cgi-bin/BLAST.

Alternatively, homology can be determined by hybridization of polynucleotides under conditions in which double-stranded molecules are formed between homologous regions, followed by digestion with specific nucleases of single chain regions and the determination of the length of digested fragments. DNA sequences that are substantially homologous can be identified by a Southern Southern hybridization experiment, e.g. eg, astringency conditions as defined for this particular system. The definition of appropriate hybridization conditions is within the field's abilities. See, p. eg, Sambrook et al , supra ; DNA cloning , supra ; Nucleic Acid Hybridization , supra .

The term "degenerate variant" refers to to a polynucleotide that contains changes in the acid sequence nuclei thereof, and encoding a polypeptide that possesses the same amino acid sequence as the polypeptide encoded by the polynucleotide from which the degenerate variant is derived.

Techniques to determine "similarity" Amino acid sequence are well known in the field. In general, "similarity" means the exact amino acid comparison to amino acid of two or more polypeptides in the appropriate places, where the amino acids are identical or have physical properties or similar chemicals, such as loading or hydrophobicity. Thus can determine what is called "percentage of similarity" between the polypeptide sequences compared. The techniques for determine the nucleic acid and amino acid sequences they also know each other well in the field and include the determination of the mRNA nucleotide sequence of that gene (usually through of a cDNA intermediary) to determine, in this way, the encoded amino acid sequence and compare this with a second amino acid sequence In general, the term "identity" refers to the exact correspondence, nucleotide to nucleotide or amino acid to amino acid, between two nucleotide sequences or polypeptide, respectively.

A "heterologous" region of a construct of DNA is an identifiable segment of DNA located within another DNA molecule, or attached to it, that naturally does not found in association with the other molecule. So when the region heterologous encodes a bacterial gene, the gene is usually flanked by DNA that does not flank that bacterial gene in the genome of the bacteria it comes from. Another example sequence heterologous coding is a construct where the sequence coding itself is not found naturally (e.g., synthetic sequences that have codons other than those of the gene native). Allelic variants or mutations that occur from naturally they do not cause the appearance of heterologous regions of DNA, as used herein.

A "vector" is a replicon, like a plasmid, a phage or a cosmid, to which a segment of DNA binds to cause replication of that joined segment. A vector is capable of transferring gene sequences to target cells (e.g., bacterial plasmid type vectors, viral vectors, non-vectors viral, particulate transporters and liposomes).

Typically, all of the following terms: "vector construct", "expression vector", "vector gene expression "," gene release vector ", "gene transfer vector" and "expression cassette"; they refer to a montage that is capable of directing the expression of a sequence or gene of interest. Thus, the term includes vehicles of cloning and expression, as well as viral vectors.

These assemblies consist of a promoter that It is functionally linked to the sequences or genes of interest. Also, other control elements may be present. The Expression cassettes described in this document may be contained in a plasmid construct. In addition to the components of the expression cassette, the plasmid construct can also include an origin of bacterial replication, one or more markers of selection, a signal that allows the plasmid construct to exist as single stranded DNA (e.g., an M13 origin of replication), a multiple cloning site and an origin of replication of "mammal" (eg, an origin of replication of an adenovirus or from SV40).

The "control elements" of the DNA refer, together, to transcription promoters, transcription stimulating elements, transcription termination sequences, polyadenylation sequences (located at the 3 'end of the translation termination codon ), sequences for optimization of translation initiation (located at the 5 'end of the coding sequence), translation termination sequences, 5' regulation domains, ribosome binding sites and the like; which, together, allow transcription and translation of a coding sequence in the host cell. See, p. eg, McCaughan et al. (1995) PNAS USA 92 : 5431-5435; or Kochetov et al. (1998) FEBS Letts. 440 : 351-355. Not all of these control sequences are always necessary in the recombinant vector, as long as transcription and translation of the gene in question is possible.

The term "functionally linked" is refers to an arrangement of the elements so that the components thus described are configured to perform their usual functions. Thus, the united control elements functionally to a coding sequence are able to lead to carry out the expression of the coding sequences. It is not necessary that the control elements are contiguous to the sequence coding, as long as they direct the expression of it. Thus, for example, between the promoter and the coding sequence intermediate transcribed sequences may exist but no translated, and the promoter can still be considered "functionally linked" to the coding sequence. So similar, "the control elements compatible with the expression in a subject "are those who are able to carry out the expression of the coding sequence in said subject.

A control element, such as a promoter, "directs the transcription" of a coding sequence in a cell when RNA polymerase binds to the promoter and transcribes the coding sequence in an mRNA, which in turn translates into the polypeptide sequence encoded in the coding sequence.

A "host cell" is a cell that has been transformed, or is capable of being, by an exogenous molecule of nucleic acids.

A cell has been "transformed" by a Exogenous DNA when said exogenous DNA is introduced into the cellular membrane. The exogenous DNA can be integrated (linked covalently) or not in the chromosomal DNA and be part of the cell genome In prokaryotes and yeasts, for example, can keep exogenous DNA in an episomic element as a plasmid As for eukaryotic cells, a cell stable transform is one in which exogenous DNA has been integrated into the chromosome, so that the daughter cells inherit through chromosomal replication. This stability is demonstrates by the ability of eukaryotic cells to establish cell lines or clones with a population of daughter cells that contain exogenous DNA.

As used herein, a "biological sample" refers to a sample of tissue or fluid isolated from a subject, which includes the following elements, although not limited to them: blood, plasma, serum, stool, urine, bone marrow, bile, cerebrospinal fluid, lymphatic fluid, skin samples, external secretions of the skin, respiratory tract, digestive tract and genitourinary system, tears, saliva, milk, blood cells, organs, biopsies and also samples of in vitro cell culture constituents that include, among others, the following elements: conditioned medium resulting from cell and tissue growth in culture medium, e.g. eg, recombinant cells and cellular components.

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As used in this document, the term "marker" or "detectable marker" refers to a molecule capable of being detected and includes the following, although without being limited to them: radioactive isotopes, molecules fluorescent, chemiluminescent molecules, enzymes, substrates enzymatic, enzymatic cofactors, enzymatic inhibitors, chromophores, dyes, metal ions, ligands (e.g., biotin or haptens) and the like. The term "fluorescent molecule" is refers to a substance or a portion thereof that is capable of emit fluorescence in a detectable range. Some examples particular of markers that can be used in this invention they are fluorescein, rhodamine, dansil, umbeliferone, Texas red, luminol, NADPH and α-? -galactosidase.

2. Ways of carrying out the invention

Before describing the present invention in detail, it should be clarified that this invention is not limited to one formulation or particular process parameters since the They may vary. It is also important to clarify that the terminology used in this document aims to describe only the particular embodiments of the invention and does not pretend to be limiting.

Although there are already a number of methods and materials similar or equivalent to those described here that they can use in the practice of the present invention, this document describes the materials and methods preferred.

A very important issue in the present invention is the discovery that the GapC protein is capable of triggering an immune response in a vertebrate subject. In particular, the genes of the GapC protein have been isolated, sequenced and characterized in S. dysgalactiae, S. agalactiae, S. uberis, S. parauberis and S. iniae , and from them the protein sequences of said genes have been deduced . The complete DNA sequences of said genes and their corresponding amino acid sequences are shown in Figures 1 through 5.

As described in the examples, the complete gapC gene of S. dysgalactiae , shown at positions 1 through 1011, both inclusive, of Figures 1A-1B, encodes the complete GapC protein of S. dysgalactiae from 336 amino acids, shown as amino acids 1 to 336 of the same figure. The S. dysgalactiae GapC protein has an expected molecular weight of about 36 kDa (calculated using the GCG Wisconsin Package Peptide Sort program, version 10, supplied with the SeqWeb sequence analysis package, version 1.1 of the Canadian Bioinformatics Resource). Similarly, the gapC genes isolated from S. agalactiae, S. uberis, S. parauberis and S. iniae are shown in Figures 2 through 5. Each of them encodes a GapC protein of 336 amino acids also with a expected molecular weight of 36 kDa. None of the sequences include a signal peptide or a membrane anchor region.

Figures 6 and 7 show the alignment of DNA or amino acid sequences, respectively, showing the regions of homology and variability between the GapC proteins from different strains of streptococci. In particular, in amino acid positions 62 to 81, 102 to 112, from 165 to 172, from 248 to 271 and from 286 to 305 are located Various variable regions. These variable regions are typically more susceptible to change. Therefore, it is more likely that changes, such as substitutions, additions and deletions, in the amino acids of these regions.

Figures 8 through 12 show the diagrams of the following characteristics for each of the GapC proteins of the present invention: hydropathy according to the scale Kyte-Doolittle (average over a window of 7); surface probability according to Emini; chain flexibility according to Karplus-Schulz; antigenicity index according Jameson-Wolf; secondary structure according Garnier-Osguthorpe-Robson; secondary structure according to Chou-Fasman; and places of glycosylation expected. The figures from 13 to 17 show Secondary structure values according to Chou-Fasman for each of the GapC proteins of The present invention. Any expert in the field may easily use the information presented in Figures 8 at 17 to determine the immunogenic regions of the protein in order to use them in the vaccine composition.

Plasma-binding GapC protein of S. dysgalactiae , immunogenic fragments thereof or chimeric proteins that include it, can be supplied as vaccine subunit compositions. In addition to their use in the vaccine composition, their proteins or antibodies can be used as diagnostic reagents to detect the presence of infection in a vertebrate subject. Similarly, the genes encoding proteins can be cloned and used to design probes that detect and isolate homologous genes in other bacterial strains. For example, fragments consisting of at least between 15 and 20 nucleotides, more preferably at least about 20 and 50 nucleotides, and even more preferably about 60 and 100 nucleotides, or any integer between those values, may be employed in these realizations

The vaccine compositions of the present invention can be used to treat or prevent a wide variety of bacterial infections in vertebrate subjects. For example, vaccine compositions containing the plasmin-binding GapC proteins of S. dysgalactiae, S. uberis, S. parauberis, S. iniae or group B streptococci (SGB) such as S. agalactiae , can be used to treat streptococcal infections in vertebrate subjects that are caused by these or other species. In particular, S. uberis and S. agalactiae are common bacterial pathogens associated with mastitis in bovids, equidae, ovines and goat species. Additionally, it is known that group B streptococci, such as S. agalactiae , cause a large number of other vertebrate infections such as septicemia, meningitis, bacteraemia, impetigo, arthritis, urinary tract infections, abscesses, spontaneous abortion, etc. Therefore, vaccine compositions containing streptococcal plasmid-binding GapC proteins will be useful in the treatment and prevention of a wide variety of streptococcal infections.

Similarly, plasmin-binding GapC proteins derived from other bacterial genera such as Staphylococcus, Mycobacterium, Escherichia, Pseudomonas, Nocardia, Pasteurella, Clostridium and Mycoplasma will be useful for the treatment of bacterial infections caused by species belonging to those genera. Thus, it is very clear that plasmin-binding GapC proteins can be used to treat or prevent a wide variety of bacterial infections caused by numerous species.

The streptococcal plasmid-binding GapC proteins of the present invention can be used in vaccine compositions independently or in combination with other bacterial, fungal, viral or protozoan antigens. These antigens can be supplied separately or as fusion proteins consisting of one or more epitopes of the plasmin-binding GapC protein fused with one or more of said antigens. For example, other S. uberis immunogenic proteins, such as the CAMP factor, the hyaluronic acid capsule, the hyaluronidase, the R-type protein and the plasminogen activator, can be administered with the GapC protein. Additionally, immunogenic proteins of other microorganisms involved in mastitis, such as those of the genus Staphylococcus, Corynebacterium, Pseudomonas, Nocardia, Clostridium, Mycobacterium, Mycoplasma, Pasteurella, Prototheca , other streptococci, coliform bacteria, as well as yeasts, can be administered together with GapC plasmin binding proteins described herein to provide a broad spectrum of protection. For example, immunogenic proteins from Staphylococcus aureus, Str. Agalactiae, Str. Dysgalactiae, Str. Zooepidemicus, Corynebacterium pyogenes, Pseudomonas aeruginosa, Nocardia asteroides, Clostridiunm perfringens, Escherichia coli, Enterobacter aerogenes and Klebsiella spp. they can be supplied together with the plasmin-binding GapC proteins of the present invention.

Additionally, GapC proteins of different stroptococcal species can be used together in vaccine compositions of the present invention. In this embodiment, multiple GapC proteins can be supplied as fusion proteins or as discrete antigens, in the same vaccine composition or in a different one.

Production of plasmin-binding GapC proteins

The plasmin binding proteins described previously and its active fragments, analogous proteins or chimeric derived from them, can be produced by a diversity of methods In particular, GapC-binding proteins plasmin can be isolated directly from the bacteria that the express. This is generally achieved by preparation, first of all, of a crude extract that has no components Cellular or several foreign proteins. The desired proteins are can subsequently purify the cell lysate fraction by, p. eg, column chromatography, HPLC, techniques immunosorbents or other conventional methods well known in the countryside.

More specifically, techniques for isolating GapC proteins from plasmin binding have been described. For example, the S. pyogenes GapC protein was purified from a crude cell extract by precipitation with ammonium sulfate followed by two cycles of chromatography through a Mono FPLC column, and single cycles through FPLC in super 12 and TSK- columns. HPLC phenol (Pancholi, V. and Fischetti, VA [1992] J. Exptl. Med. 76 : 415-426). There is another technique that involves the use of NAD +-agarose affinity columns to purify GapC from lysate protoplasts of S. pyogenes strain 64/14 (Winram, SB and Lottenberg, R [1996] Microbiol. 142 : 2311 -2320).

Alternatively, proteins can be produced recombinantly as described herein. As explained above, these recombinant products can take the form of partial protein sequences, complete sequences, precursor forms that include signal sequences, mature forms without signal sequences, or even fusion proteins (e.g., with a sequence appropriate leader for the recombinant host or with sequences of antigenic subunits of Streptococcus or another pathogen).

In one embodiment of the present invention, GapC proteins are fused with a histidine tail produced by recombinant methods and then purified from the cell lysate fraction using affinity chromatography. See Example 1A-E, infra .

The plasmin-binding GapC proteins of the present invention can be isolated, thanks to the ability of the protein product to bind plasmin, by the plasmin binding assays described below. See, p. eg, the method described in section F.3 of Example 1, infra . Thus, libraries can be constructed and the resulting clones can be used to transform the appropriate host cells. Colonies can be pooled and screened for clones that possess plasmin binding activity. Colonies can also be screened using polyclonal sera or monoclonal antibodies against plasmin binding protein.

Alternatively, once the amino acid sequences have been determined, oligonucleotide probes containing the codons of a portion of the determined amino acid sequence can be used to screen cDNA libraries or genomic DNA libraries and search for protein genes in question. The basic strategies for the preparation of oligonucleotide and library probes, as well as for their screening by nucleic acid hybridization, are techniques that are well known to those skilled in the art. See, p. eg, DNA Cloning : Vol. 11, supra; Nucleic Acid Hybridization, supra; Oligonucleotide Synthesis, supra ; Sambrook et al. supra Once a clone of the screened library has been identified by positive hybridization, it can be confirmed that that particular insert of the library contains the plasmin-binding GapC protein gene or a homologue thereof by restriction enzyme analysis and DNA sequencing The genes can be subsequently isolated using standard techniques and, if desired, PCR or restriction enzyme techniques used to remove portions of the entire sequence.

Similarly, genes can be isolated directly from bacteria using known techniques, such as phenol extraction, and then the sequence can be manipulated to produce any desired alteration. See, p. eg, Sambrook et al. , supra , for a description of the techniques used in DNA isolation.

Alternatively, the DNA sequences encoding the proteins of interest can be prepared synthetically instead of cloning them. DNA sequences can be designed with the appropriate codons for each particular amino acid sequence. In general, preferred codons for host cells are selected if the sequence is to be used for expression. The complete sequence is composed of overlapping oligonucleotides prepared by standard and combined methods to form a complete coding sequence. See, p. eg, Edge (1981) Nature 292 : 756; Nambair et al. (1984) Science 223 : 1299; or Jay et al. (1984) J. Biol. Chem. 259 : 6311.

Once the coding sequences for the desired proteins have been prepared or isolated, they can be cloned into any appropriate vector or replicon. Those skilled in the art know numerous cloning vectors and the selection of an appropriate cloning vector is a matter of personal choice. Examples of vectors for cloning recombinant DNA, and host cells they can transform, include the following: bacteriophage \ lambda (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT23O (gram-negative bacteria), pGV1106 (gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290 (gram-negative bacteria that are not E. coli ), phv14 ( E. coli and Bacillus subtilis ), pBD9 ( Bacillus ), pIJ61 ( Streptomyces ), pUC6 ( Streptomyces ), YIp5 ( Saccharomyces ), YCp19 ( Saccharomyces ) and bovine papillomavirus (mammalian cells). See, Sambrook et al. , supra ; DNA Cloning, supra ; or B. Perbal, supra .

The gene can be placed under the control of a promoter, a ribosome binding site (for expression in bacteria) and, optionally, an operator (in this document, all together refer to "control" elements), of such that the DNA sequence encoding the protein of interest is transcribed into an RNA in the host cell transformed by a vector containing this protein expression construct. The coding sequence may contain a signal peptide and a leader sequence, or not contain them. If a signal sequence is included, this may be the homologous native sequence or a heterologous sequence. For example, the signal sequence of the plasmid-binding GapC protein of S. dysgalactiae can be used for secretion thereof, as well as a number of other sequences well known in the field. The host can eliminate the leader sequences during post-translational processing. See, p. eg, U.S. Patent Nos. 4 431 739, 4 425 437 and 4 338 397.

Other regulatory sequences can also be of interest when they allow the regulation of the expression of the protein sequence as a function of host cell growth. Those skilled in the art know these regulatory sequences well. and among the examples of these sequences are those that activate or inhibit the expression of a gene in response to a stimulus chemical or physical, including the presence of a compound regulator. Other types may also be present in the vector. of regulatory elements, for example, sequences Stimulators

Control sequences and other sequences regulators can join the coding sequence before their insertion into the vector, as described cloning vectors previously. Alternatively, the coding sequence can clone directly into an expression vector that already contains the control sequences and an appropriate restriction site.

In some cases, it may be necessary to modify the coding sequence so that they bind to the control sequences with the appropriate orientation, that is, to maintain the appropriate reading pattern. It would also be desirable to produce mutants or analogs of the plasmin-binding GapC protein. It is possible to prepare mutants or analogs by deleting a portion of the sequence encoding the protein, by inserting a sequence or by replacing one or more nucleotides within the same sequence. Nucleotide sequence modification techniques, such as directed mutagenesis, are described, for example, in Sambrook et al. , supra ; DNA Cloning, supra ; Nucleic Acid Hybridization , supra .

The expression vector is then used to transform an appropriate host cell. A number of mammalian cell lines that include immortalized cell lines available in the American Type Culture Collection (ATCC) are known in the field, some of which are, among others, Chinese hamster ovary (CHO) cells, HeLa cells , hamster calf kidney cells (BHK), monkey kidney cells (COS), human hepatocellular carcinoma cells (eg Hep G2), Madin-Darby bovine kidney cells ("MDBK") and the like. Similarly, host bacterial cells such as E. coli , Bacillus subtilis and Streptococcus spp. , may also be used with the present expression constructs. Host yeasts useful in the present invention include, among others, Saccharomyces cerevisiae, Candida albicans, Candida maltose, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica . Insect cells that can be used with baculovirus expression vectors include, among others, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda and
Trichoplusia ni .

Depending on the expression system and the chosen host, the proteins of the present invention are produced by culturing host cells transformed by an expression vector described above under the conditions in which the protein of interest is expressed. Then it is isolated the protein of the host cells and is purified. If the system secret expression of the protein in the culture medium, the protein It can be purified directly from the medium. If the protein is not secreted, it is isolated from cell lysates. The selection of culture conditions and appropriate recovery methods are found within the field techniques.

The proteins of the present invention can also be produced by chemical synthesis, such as solid phase peptide synthesis, using known amino acid sequences or amino acid sequences derived from the DNA sequence of the genes of interest. These methods are known to those skilled in the art. See, p. eg, JM Stewart and JD Young, Solid Phase Peptide Synthesis , 2nd Ed., Pierce Chemical Co., Rockford, IL (1984) and G. Barany and RB Merrifield, The Peptides: Analysis, Synthesis, Biology , editors E. Gross and J. Meienhofer, Vol. 2, Academic Press, New York, (1980), pp. 3-254, for solid phase peptide synthesis techniques; and M. Bodansky, Principles of Peptide Synthesis , Springer-Verlag, Berlin (1984) and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, supra , Vol. 1, for classical synthesis in solution . The chemical synthesis of peptides may be preferable if a small fragment of the antigen in question has the ability to elicit an immune response in the subject of interest.

The plasmin-binding GapC proteins of the present invention, or their fragments, can be used to produce antibodies, both polyclonal and monoclonal. If polyclonal ones are chosen, a selected mammal (eg, a mouse, rabbit, goat, horse, etc.) is immunized with an antigen of the present invention, a fragment thereof or a mutated antigen. Serum is collected from the immunized animal and treated according to known procedures. See, p. ex. Jurgens et al. (1985) J. Chrom. 384 : 363-370. If serum containing polyclonal antibodies is used, polyclonal antibodies can be purified by immunoaffinity chromatography using standard procedures.

One skilled in the art can also easily produce monoclonal antibodies against GapC plasmin-binding proteins and fragments thereof. The general methodology for producing monoclonal antibodies using hybridoma technology is widely known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA or transfection with Epstein-Barr virus. See, p. eg, M. Schreier et al. , Hybridoma Techniques (1980); Hammerling et al. , Monoclonal Antibodies and T-cell Hybridomas (1981); Kennett et al. , Monoclonal Antibodies (1980); see also US Patents no. 4,341,761; 4 399 121; 4 427 783; 4,444,887; 4 452 570; 4 466 917; 4 472 500, 4 491 632; and 4,493,890. Monoclonal antibody profiles produced against the plasmin-binding GapC proteins, or fragments thereof, can be analyzed for various properties; that is, isotype, epitope, affinity, etc. Monoclonal antibodies serve for the purification, by immunoaffinity techniques, of the individual antigens against which they are directed. Both polyclonal and monoclonal antibodies can also be used for passive immunization or can be combined with preparations with vaccine subunits to enhance the immune response. Polyclonal and monoclonal antibodies also serve diagnostic purposes.

Vaccine Formulations and Administration

The plasmin-binding GapC proteins of the present invention can be formulated in the form of vaccine compositions, individually or in combination with other antigens, for use in the immunization of subjects described below. Remington's Pharmaceutical Sciences , Mack Publishing Company, Easton, Pennsylvania, 18th Edition, 1990, for example, describes methods for preparing formulations of this type. Normally the vaccines of the present invention are prepared in injectable form, as liquid solutions or suspensions. Solid forms suitable for solution or suspension in liquid vehicles prior to injection can also be prepared. The preparation can also be emulsified or the active ingredient encapsulated in liposomal vehicles. The active immunogenic ingredient is generally mixed with a compatible pharmaceutical carrier, such as water, saline, dextrose, glycerol, ethanol or the like, or with combinations thereof. In addition, if desired, the vehicle may contain smaller amounts of adjuvant substances such as wetting or emulsifying agents and pH buffering agents.

The adjuvants that enhance the effectiveness of The vaccine can also be added to the formulation. These can be, for example: muramyl dipeptides, avridine, hydroxide aluminum, dimethyldioctadecyl ammonium bromide (DDA), oils, oil emulsions in water, saponins, cytokines or others substances known in the field.

The plasmin binding protein may be attached to a transporter in order to increase immunogenicity Of the same. Suitable transporters are large molecules of Slow metabolism like proteins. These include: albumins serum, California limpet hemocyanin, molecules of immunoglobulin, thyroglobulin, ovalbumin and other proteins well known to those skilled in the art; polysaccharides like the sepharose, agarose, cellulose, cellulose microspheres and the like; polymeric amino acids such as glutamic acid, polylysine and Similar; amino acid copolymers; and viral particles inactive

GapC plasmin binding proteins can be used in its native form or the content can be modified of its functional group by, for example, succinylation of resin residues or by reaction with Cys-thiolactone. It is also possible to incorporate in the transporter (or antigen) a sulfhydryl group by, by example, the reaction of amino functions with 2-iminothiolane or the N-hydroxysuccinimide ester of propionate 3- (4-Dithiopyridyl propionate. You can also modify the appropriate conveyors to incorporate branches spacers (such as hexamethylene diamine or other molecules bifunctional of similar size) that allow the union of the peptides

Other conveyors suitable for GapC plasmin binding proteins of the present invention include  VP6 polypeptides of rotavirus, or functional fragments of same, which are described in US Patent No. 5,071 651. It is also possible to use a fusion product of a viral protein and the subject's immunogens, carried out by methods that are described in US Pat. no. 4 722 840. Still other carriers include cells such as lymphocytes, since this form of presentation mimics the natural way of presentation in the subject, which generates the immunized state. Alternatively, the proteins of the present invention can be coupled to erythrocytes, preferably erythrocytes subject's own. Those skilled in the art know the methods of coupling peptides to proteins or cells.

In addition, plasmin binding GapC proteins (or complexes thereof) can be formulated in compositions of vaccines in their natural form or in the form of salt. Salts Pharmaceutically acceptable include acid addition salts (formed with the free amino groups of the active polypeptides) which are formed with inorganic acids such as acids hydrochloric or phosphoric, or organic acids such as acetic acid, oxalic, tartaric, mandelic and the like. The salts formed to from free carboxyl groups they can also come from bases inorganic, such as sodium, potassium, ammonium hydroxides, calcium or ferric, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, Procaine and the like.

Vaccine formulations must contain a "therapeutically effective amount" of the ingredient asset, which is an amount sufficient to provide a immune response in the subject to whom the composition. In the treatment and prevention of mastitis, by example, a "therapeutically effective amount" would be preferably an amount that improves the strength of the mammary gland to a new infection or to reduce the severity disease clinic. This protection will be demonstrated by a reduction or lack of symptoms normally shown by a infected host, for a shorter recovery time or for a lower somatic cell count in fourth milk infected. As an example, the ability of the composition to keep somatic cell count (RCS) in milk in a 500,000 cells per ml or take it to that figure, the threshold value determined by the International Dairy Federation above which animals are considered to have clinical mastitis, It should be indicative of a therapeutic effect.

The exact amount can be determined by an expert in the matter using standard tests. The concentration of GapC plasmin binding protein usually ranges from 1 to 95% (p / p) of the composition, or even higher or lower values in the cases deemed necessary. With the present formulations for vaccines, 5 to 500 µg of active ingredient per ml of solution infected should be sufficient to generate a response immune when a dose of 1 to 3 ml has been administered per animal.

To immunize a subject, the vaccine is generally administered parenterally, usually by intramuscular injection. However, other modes of administration, such as subcutaneous, intraperitoneal and intramuscular injection, They are also acceptable. The amount to be administered depends on the animal to deal with, the ability of the animal's immune system to synthesize antibodies and the degree of protection desired. The dose effective can be established by any expert in the field through routine tests establishing response curves to the dose. Immunization of the subject is performed by administering the vaccine. in one dose at least, and preferably in two. In addition, to the animal You may be given as many doses as necessary to maintain a state of immunity against infection.

Other additional formulations suitable for other modes of administration include suppositories and, in some cases, aerosols, intranasal or oral administration formulations, and controlled release formulations. In the case of suppositories, the composition of the vehicle will include binders traditional and transporters, such as polyalkaline glycols or triglycerides These suppositories can be formed from mixtures containing the active ingredient in an approximate range between 0.5 and 10% (w / w) or, preferably, between 1 and 1 2%. Oral vehicles include excipients used usually as are, for example, pharmaceutically amounts Desirable of: mannitol, lactose, starch, magnesium stearate, cellulose, sodium saccharin, magnesium carbonate and the like. These oral vaccine compositions can be administered in the form of solutions, suspensions, tablets, pills, capsules, controlled release formulations or powders, and may contain between 10 and 95% of the active ingredient, preferably between 25 and 70%.

Intranasal formulations must include normally vehicles that do not cause irritation in the nasal mucosa and that do not significantly alter the ciliary function. With the present invention it is possible to use diluents such as water, aqueous saline solution or other known substances. The Nasal formulations may also contain preservatives such as, for example, chlorobutanol and benzalkonium chloride. Can it be added a surfactant to improve the absorption of proteins in issue for the nasal mucosa.

The controlled release formulations or Sustained are produced by incorporating protein into transporters or vehicles such as the following: liposomes, non-absorbable polymers waterproof as copolymers of ethylene vinyl acetate and Hytrel® copolymers, swellable polymers such as hydrogels, or resorbable polymers such as collagen and certain polyacids or polyesters such as those used to make absorbable sutures.  GapC plasmin binding proteins can also be administer by widely used implanted mini pumps in the countryside.

The plasmin-binding GapC proteins of the invention that concerns us can also be administered through a transporter virus that expresses these proteins. The virus conveyors that are useful in the present invention they include, among others, vaccinia virus and other smallpox viruses, adenovirus and herpes virus. As an example, you can build recombinant vaccinia virus that express the new proteins as follows. The DNA that encodes the protein in particular it is inserted first so that it is adjacent to a vaccinia promoter and vaccinia DNA sequences flanking agents, such as the sequence encoding thymidine kinase (TK) This vector is then used to transfect cells. which are infected simultaneously with vaccinia. The homologous recombination serves to insert the vaccinia promoter plus the gene that encodes the protein in question within the genome viral. The resulting TK recombinant can be selected by culturing the cells in the presence of 5-Bromodeoxyuridine and bald selection viral resistant to it.

An alternative route of administration involves gene therapy or genetic immunization. Therefore, the nucleotide sequences (and the accompanying regulatory elements) encoding the plasmid-binding GapC proteins of interest can be directly administered to a subject for in vivo translation thereof. In contrast, gene transfer can be performed by transfecting the cells or tissues of the ex vivo subject and reintroducing the transformed material into the host. DNA can be introduced directly into the host microorganism, that is, by injection (see International Publication No. WO / 90/11092; and Wolff et al. [1990] Science 247 : 1465-1468). Liposome-mediated gene transfer can also be achieved using known methods. See, for example, Hazinski et al. (1991) Am. J. Respir. Cell Mol. Biol. 4 : 206-209; Brigham et al. (1989) Am. J. Med. Sci. 298 : 278-281; Canonico et al. (1991) Clin. Res. 39 : 219A; and Nabel et al. (1990) Science 249 : 1285-1288. Targeted agents, such as antibodies directed to surface antigens expressed in specific cell types, can be covalently conjugated to the liposomal surface so that the nucleic acid can be released into specific tissues and cells vulnerable to infection.

Diagnostic tests

As explained above, the plasmin-binding GapC proteins of the present invention can also be used as a diagnostic means to detect the presence of streptococcal reactive antibodies, for example to S. dysgalactiae , in a biological sample for the purpose of determining the existence of streptococcal infection. For example, the presence of antibodies reactive to plasmid-binding GapC proteins can be detected by standard electrophoresis and immunodiagnostic techniques, including immunoassays such as competitive-type, direct-reaction or sandwich-type assays. These tests include, among others: Western blots, agglutination assays, enzyme-labeled or enzyme-mediated immunoassays (such as ELISA), biotin / avidin-type assays, radioimmunoassays, immunoelectrophoresis, immunoprecipitation, etc. The reactions generally include the manifestation of fluorescent, chemiluminescent, radioactive or enzymatic labels, or staining molecules, or other methods to detect the formation of a complex between the antigen and the antibody or antibodies with which it reacts.

These mentioned tests generally involve the separation of the unbound antibody in a liquid phase from a solid phase support to which the antigen-antibody complexes are attached. Solid supports that can be used in the practice of the invention include substrates such as nitrocellulose (eg in the form of a membrane or microtiter well), polyvinyl chloride (eg sheets or microtiter wells), latex of polystyrene (eg microspheres or microtiter plates), polyvinylidine fluoride, diazotized paper, nylon membranes, activated microspheres, magnetic attraction microspheres and the like.

Typically, a solid support is first reacted with a solid phase component (eg one or more plasmin-binding GapC protein) under suitable binding conditions so that the component is sufficiently fixed to the support. Sometimes the binding of the antigen to the support can be enhanced by first coupling the antigen to a protein with better binding properties. Some of the suitable coupling proteins are macromolecules of the type: serum albumin such as bovine serum albumin (BSA), Californian lapa hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the field. Other molecules that can be used to bind the antigens to the support include polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and the like. These molecules and methods of coupling these molecules to the antigens are well known to any expert in the field. See, p. eg, Brinkley, MA Bioconjugate Chem. (1992) 3 : 2-13; Hashida et al. , J. Appl. Biochem (1984) 6 : 56-63; and Anjaneyulu and Staros, International J. of Peptide and Protein Res. (1987) 30 : 117-124.

Once the solid support has been reacted with the solid phase component, it is removed by washing any solid phase component not immobilized on the support, and the component attached to the support is then contacted with a biological sample suspected of containing fractions of the ligand (eg antibodies against antigens immobilized) under suitable binding conditions. After the wash to remove any unbound ligand, a secondary binding fraction under the appropriate conditions of binding, so that the secondary binder is able to associate with Selectively with bound ligand. The presence of the linker secondary can then be detected using the techniques widely known in the field.

More specifically, you can use a ELISA method in which the wells of a microtiter plate they are coated with a plasmin-binding GapC protein. TO a sample is then added to the coated wells biological that it contains, or suspected of containing, GapC anti-protein immunoglobulin molecules of plasmin binding. After a sufficient incubation period to allow the binding of the antibody to the immobilized antigen, the plate or plates can be washed to remove unbound fractions and a shapely labeled secondary binding molecule can be added detectable The secondary binding molecule is allowed to react with any antibody captured from the samples, the plate is washed and the presence of the secondary binding molecule is detected by surplus methods known in the field.

Therefore, in a particular embodiment, the presence of bound antigen ligands anti-plasmid binding GapC protein from of a biological sample can be easily detected using a secondary linker containing an antibody directed against antibody ligands. In the field a series of immunoglobulin (Ig) antibovin molecules that allow a rapid conjugation with a detectable enzyme marker, such as Horseradish peroxidase, alkaline phosphatase or urease, using methods known to those skilled in the art. A appropriate enzyme substrate is used below to Generate a detectable signal. Other related realizations support the use of competitive ELISA techniques using the surplus methods known to experts.

The tests can also be carried out in solution, so that specific proteins and antibodies for these proteins form complexes under conditions of precipitation. In a specific embodiment, GapC proteins Plasmin binding can bind to a solid phase particle (e.g. ex. an agarose microsphere or similar) using the techniques Standard coupling, such as direct chemical coupling or indirect. Then the coated particle is contacted with antigens under binding conditions with a biological sample of the suspected to contain antibodies to GapC proteins from plasmin binding. Cross linking between bound antibodies causes complex aggregate formation particle-antigen-antibody that they can be precipitated and separated from the sample by washing or centrifuged The reaction mixture can be analyzed for determine the presence or absence of complexes antibody-antigen using any of the several standard methods, such as immunodiagnostic methods described above.

In yet another embodiment, it can be provided an immunoaffinity matrix in which it is immobilized in a substrate a polyclonal population of antibodies from a sample biological suspect to contain molecules anti-GapC plasmin binding. In that sense, it is possible to carry out an initial purification of the sample by affinity with immobilized antigens. In this way, the preparation of resulting samples will only contain fractions anti-streptococci, thus avoiding possible binding characteristics unspecific in affinity support. In the field they know each other various immunoglobulin immobilization methods (intact or in specific fragments) with high performance and good retention of antigen binding activity. Without being limited by any particular method, protein A or G protein to immobilize immunoglobulins.

Consequently, once they are immobilized immunoglobulin molecules to provide a matrix of immunoaffinity, the GapC binding proteins are contacted plasmin labeled with antibodies bound under conditions of adequate union. Once they have washed the immunoaffinity support all antibodies bound nonspecifically, you can determine the presence of bound antigen by assessing the presence of the marker by known methods.

In addition, the antibodies generated against the GapC plasmin binding proteins, better than the proteins themselves Plasmin binding GapC, can be used in assays before described in order to detect the presence of antibodies to the proteins in a given sample. These tests are performed fundamentally in the manner described above and are of general knowledge in the field.

The reagents of the tests described above, including GapC plasmin binding proteins or antibodies of these, can be supplied in equipment that carry the proper instructions and other reagents necessary to carry perform the immunoassays described above. The team can also contain, depending on the immunoassay used in each case, suitable markers and other reagents and packaged materials (en say, wash buffers and the like). Standard immunoassays as described above can be done using these equipment.

Deposits of the strains useful for the practice of invention

A crop deposit was made Biologically pure of the following strains in the Collection Type Crop American, 10801 University Boulevard, Manassas, Virginia, under the guidelines of the Budapest Treaty. The number of indicated access was assigned after a positive analysis of viability, and the required fees were paid. Deposits Designated will be maintained for a period of thirty (30) years from the date of deposit, or for five (5) years after the Last request for deposit, the period of time that is longer long. In the event that a crop is unfeasible or destroyed from unintentionally, or strains containing plasmids the lose, will be replaced by a viable crop or crops of the Same taxonomic description.

If there were a discrepancy between the sequence presented in the present application and the gene sequence of plasmid interest deposited due to routine errors of sequencing, the sequence of the deposited plasmid will be the one prevail

Bacterial strain Plasmid Gen Date Deposit no. ATCC E. coli BL2I DE3 pET15bgapC gapC ( S. dysgalactiae ) May 31 2000 PTA-1976 E. coli BL21 DE3 pMF521c gapC ( S. agalactiae ) May 31 2000 PTA-1975 E. coli BL21 DE3 pMF521a gapC ( S. uberis ) May 31 2000 PTA-1973 E. coli BL21 DE3 pMF521e gapC ( S. iniae ) May 31 2000 PTA-1972

3. Experimental

The following are examples of specific embodiments for the realization of this invention. The examples are offered for one purpose only. illustrative and are not intended to limit the scope of this invention in any way.

Efforts have been devoted to ensuring the accuracy with respect to the numbers used (eg quantities, temperatures, etc.), but it is possible that there are some experimental deviation or error.

Materials and methods

Enzymes were purchased from sources commercial and were used following the instructions of the Manufacturers

For the isolation of DNA fragments, except in cases where otherwise specified, all DNA manipulations were performed according to standard procedures. See Sambrook et al. supra The restriction enzymes T4 DNA ligase, E. coli , DNA polymerase II, Klenow fragment and other biological reagents can be purchased from commercial suppliers and used in accordance with the manufacturer's instructions. The double stranded DNA fragments were separated on agarose gels.

Sources of chemical reagents include in General the following: Sigma Chemical Company, St. Louis, MO; Alrich, Milwaukee, WI; Roche Molecular Biochemicals, Indianapolis, IN.

Example 1 Preparation, amplification, sequencing, expression, purification and characterization of S. dysgalactiae plasmin binding GapC protein . A. Preparation of the chromosomal DNA of S. dysgalactiae

A clinical isolate of S. dysgalactiae from a case of bovine mastitis (ATCC Access No. ATCC43078) was obtained from the American Collection of (10801 University Boulevard, Manassas, VA 20110-2209), and was used as a source of DNA. The microorganism was routinely cultured on TSA agar plates and bovine blood (PML Microbiologicals, Mississauga, Ontario) at 37 ° C for 18 hours, or in Tod & Hewitt broth (Oxoid Ltd., Hampshire, England) supplemented with 0.3% of yeast extract (THB-YE) at 37 ° C and 5% CO2.

The chromosomal DNA was prepared from S. dysgalactiae grown in 100 ml of THB-YE supplemented with 20 mM glycine for approximately 6 hours, until an A 600 of 0.8 to 1.0 was reached. Cells were collected and resuspended in 50 mM EDTA, 50 mM Tris-HCl and 0.5% Tween-20® (Sigma, St. Louis, MO), and supplemented with ribonuclease A (200 mg / ml), Proteinase K (20 mg / ml), lysozyme (100 mg / ml) and mutanolisin (100 mg / ml) (SIGMA, St. Louis, MO). After bacterial lysis for 30 minutes at 37 ° C with vigorous stirring, guanidine hydrochloride and Tween-20® were mixed at pH 5.5 with the lysate to achieve a final concentration of 0.8 M and 5%, respectively. This mixture was incubated at 50 ° C for 30 minutes. The chromosomal DNA was then purified using a 100 g Qiagen genomic-tip kit (Qiagen, Santa Clarita, CA) and precipitated using 0.7 volumes of isopropanol. The resulting precipitate was washed in 70% ethanol and resuspended in 0.5 ml of 10 mM Tris-HCl at pH 8.8.

B. Amplification and cloning of the gapC gene of S. dysgalactiae

The gapC gene was amplified by PCR (see Mullis et al, U.S. Patent No. 4,683,195,.. Mullis, US Patent No. 4,683,202.). The direct primer, gapC1 , contained a NdeI restriction target (SEQ ID NO : 1, shown in Table 1) and the indirect primer, gapC1r , contained a BamHI target (SEQ ID NO : 2 , shown in Table 1). In the previous primer sequences, illustrated in Table 1, the underline indicates the nucleotides added to the original sequence, and the bold type indicates the location of restriction endonuclease recognition sites.

PCR was carried out using Vent DNA polymerase (New England Biolabs, Mississauga, ON, Canada). 0.7 µg of S. dysgalactiae chromosomal DNA was incubated in a reaction mixture containing 1 µM of each of the above primers; 200 µM of each of the nucleotides dATP, dTTP, dCTP and dGTP; 3 mM MgSO 4; 1x concentration of Thermopol buffer (New England Biolabs, Mississauga, ON, Canada); and 2 units of Vent DNA polymerase. This mixture was incubated for 3 amplification cycles of 1 minute at 94 ° C, 3 minutes at 50 ° C and 1 minute, 10 seconds at 72 ° C, then for 27 amplification cycles of 15 seconds at 95 ° C, 30 seconds at 55 ° C, and 1 minute at 72 ° C , and finally for 1 cycle of 5 minutes at 72 ° C.

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TABLE 1 Sequence and sequence identification numbers corresponding amino acids and nucleotides

one

2

The gapC PCR product was cloned into an expression vector pET15B (Novagen, Madison, WI) that had been digested with BamHI and NdeI . Cloning of the PCR product at this site causes the addition of a coding sequence into the reading pattern for a tail of six histidines to the gapC coding sequence. Subsequent expression produces a complete protein that carries the histidine tail, which allows purification of the protein under non-denaturing conditions using metal chelate chromatography.

This construct was used to transform E. coli BL21 DE3 (Life Technologies, Gaithersburg, MD). This transformed strain was designated BL21 DE3 (PETl5bgapC) (No. ATCC).

C. Isolation of chromosomal DNA and amplification and cloning of the gapC gene of S. agalactiae , S. uberis , S. parauberis and S. iniae

The gapC gene was prepared from other isolates essentially following the manner described above.

The chromosomal DNA of S. agalactiae , S. uberis and S. parauberis was isolated from strains obtained from the American Type Culture Collection (10801 University Boulevard, Manassas, VA 20110-2209; strains designated by ATCC 27541, 9927 and 13386, respectively ). The chromosomal DNA of S. initie was isolated from a strain designated 9117 obtained from Mount Sinai Hospital in Toronto.

The primers used to amplify the gapC genes of the Streptococcus strains described above were the same as those used in the case of S. dysgalactiae , that is, the gapC1 primer (SEQ ID NO : 1) and the gapC1r primer (SEQ ID No. 2).

Once the amplification was finished, the PCR product was cloned in each case into a pPCR-Script, using the cloning protocol described in the PCR-Script Amp Cloning Kit (Stratagene, La Jolla, CA). Then cut the PCR product insert using NdeI and BamHI and was recloned into those sites in pEl5b using conventional cloning protocols (see, e. G., Sambrook et al., Supra.). Plasmids containing S. agalactiae, S. uberis, S. parauberis and S. iniae were designated as pMF521c (ATCC No.), pMF521a (ATCC No.), pMF521d (ATCC No.) and pMF521e (ATCC No. ), respectively.

D. Nucleotide sequence of the gapC gene and deduced amino acid sequences

Sequencing a gene of S. dysgalactiae bound but not related was when the homologous sequences to gapC of the homologue of S. equisimilis were first identified (Gase, et al. [1996] European J. of Biochem. 239 : 42-51 ). To obtain the complete sequence of the gapC gene for S. dysgalactiae, PCR was employed with the primers described above, i.e., primer gapC1 and the primer gapC1r.

The sequence was determined in the Plant Biotechnology Institute (PBI, Saskatoon, Canada) using fluorescently labeled tail terminators in a sequencer of automatic DNA ABI 373 (Applied Biosystems, Emeryville, CA).

The sequences of GapC proteins isolated from S. agalactiae , S. uberis , S. parauberis and S. iniae were determined by the same method.

Figures 1 through 5 show both the nucleotide sequences and the expected amino acid sequences of the S. dysgalactiae GapC protein (DysgalGapC) (SEQ ID NO: 3 and SEQ ID NO: 4) as well as for the GapC proteins of S. agalactiae (AgalGapC) (SEQ. ID. No .: 5 and SEQ. ID. No .: 6), S. uberis (UberGapC) (SEQ. ID. No. .: 7 and SEC ID No.: 8), S. parauberis (PUberGapC) (SEC ID No. 9: and SEQ ID No. 10) and S. iniae (IniaeGapC) ( SEC ID No. 11 and SEC ID No. 12), respectively.

Gene GapC protein S. dysgalactiae of depicted in Figures 1A-1B encoding a protein of 336 amino acids not appear to contain a signal sequence or a domain or membrane anchor. A search in the GenBank database conducted with the BLASTX program revealed that the open reading pattern was 95.5% homologous to the S. equisimilis GapC (GenBank accession number X97788) and in 99, 4% to the GapC of S. pyogenes (Accession no. In GenBank M95569). The predicted amino acid sequence of the GapC protein also showed a 43% amino acid identity with bovine glyceraldehyde-3-phosphate dehydrogenase (Accession No. in GenBank U85042).

Similarly, no presence of signal sequences or membrane anchor domains is observed in the GapC protein sequences of S. agalactiae, S. uberis, S. parauberis and S. iniae .

The sequence homologies appear represented in Table 2.

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TABLE 2 Sequence homologies between different sequences of the GapC protein

3

E. Expression and purification of S. dysgalactiae recombinant GapC binding protein.

GapC protein with a polyhistidine tail (6 His) was expressed and purified under non-denaturing conditions by affinity chromatography of metal chelates (Ni-NTA).

E. coli BL21 DE3 containing the recombinant plasmid was grown in Luria Broth medium (LB) containing 100 µg / ml ampicillin to an A 600 of about 0.5. Then, GapC protein expression was induced by the addition of 1 mM isopropyl-? D-thiogalactoside (IPTG) (Sigma, St. Louis, MO). After three hours of incubation at 37 ° C, the cells were collected, washed with the column buffer (50 mM sodium phosphate solution, pH 8.0; 0.3 M NaCl; 10 mM imidazole) and lysed by sonication.

Approximately 40% of the recombinant protein was in the soluble fraction of the cell lysate with an approximate yield of 50 mg of recombinant protein per liter of culture, determined by the DC Protein Valuation Equipment (Bio-Rad Laboratories, Mississauga, ON , Canada) using bovine serum albumin (Pierce, Rockford, IL) as standard.

The lysate was cleaned by centrifugation and the soluble fraction was passed through a Ni-NTA column (Qiagen), which was subsequently washed with 10 column volumes of column buffer (same as above, except that it contained 20 mM imidazole). The GapC protein with the polyhistidine tail is eluted using the column buffer (same as above, except that it contained 250 mM imidazole) and resulted in a homogeneous protein fraction with a GapC concentration of 10 at 15 mg / ml. This fraction was dialyzed against 2000 volumes of PBSA (136 mM sodium chloride, 2.6 mM potassium chloride, sodium phosphate 8.1 mM dibasic, 1.46 mM monobasic potassium phosphate).

E. Expression and purification of the recombinant plasmid-binding GapC protein from S. agalactiae , S. uberis , S. parauberis and S. iniae

The expression and purification of the recombinant proteins of these streptococcal species is achieved by the same procedures described in Example 1E mentioned above. Transformed bacterial strains used to express the recombinant GapC proteins of S. agalactiae , S. uberis , S. parauberis and S. iniae were designated BL21 DE3 (pMF52lc) (ATCC No.), BL21 DE3 (pMF521a) (ATCC No. ), BL21 DE3 (pMF52ld) (ATCC No.) and BL21 DE3 (pMF521e) (ATCC No.), respectively.

G. Characterization of the recombinant GapC protein of S. dysgalactiae 1. Gel analysis of polyacrylamide-SDS

Polyacrylamide-SDS gel electrophoresis was performed with a sample of the eluted protein by the method described by Laemli (Laemli, UK [1970] Nature 227: 680-685). The results are presented in Figure 18: lane 1, molecular weight markers (range from 20.5 to 103 kDa; BioRad Laboratories, Emeryville, CA); lane 2, soluble recombinant GapC protein of S. dysgalactiae , purified by Ni-NTA affinity chromatography.

The results show that purification by Ni-NTA column affinity chromatography gives as resulted in a homogeneous protein fraction.

2. Gliceraldehyde-3-phosphate dehydrogenase (GAPDH) activity of the GapC recombinant protein and S. dysgalactiae whole cells

GAPDH enzyme catalyzes phosphorylation oxidative of D-glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate in the presence of NAD + and inorganic phosphate The high degree of homology of GapC with the glyceraldehyde-3-phosphate streptococcal dehydrogenase suggests that GapC could have that enzymatic activity.

The GAPDH activity of the entire S. dysgalactiae cells (10 10 CFU) and of the recombinant GapC protein was determined by measuring the reduction of NAD + to NADH. The assay buffer was composed of 40 mM triethanolamine, 50 mM Na 2 HPO 4 and 5 mM EDTA, pH 6.8. S. dysgalactiae cells or 5 mg of purified GapC recombinant protein were incubated in the assay buffer containing glyceraldehyde-3-phosphate (49 mg / ml; Sigma Chemical Company), 75 microliters of NAD + (15 mM; Sigma Chemical Company) to a final volume of 1 ml. Negative controls were samples that did not contain glyceraldehyde-3-phosphate or recombinant GapC protein molecules / S. dysgalactiae cells. The reduction of NAD + to NADH was monitored spectrophotometrically at an absorbance of 340 nanometers.

The results indicated that both the recombinant protein and the intact cells of wild-type S. dysgalactiae possessed enzymatic activity (these results are not shown). In addition, when S. dysgalactiae cells were treated with trypsin to digest surface proteins, GAPDH activity disappeared. Thus, the enzymatic activity observed in intact wild-type cells was not due to intracellular GAPDH.

These data suggest that the GapC protein is locates on the surface of the cell despite the apparent lack of signal or membrane anchor sequences both in the sequence of nucleotides as in amino acids.

3. Plasmin - Binding Activity of Recombinant GapC plasmin binding protein from S. dysgalactiae and whole cells of S. dysgalactiae

A microplate assay was used to determine if the recombinant GapC protein had the ability to bind to bovine plasmin, and if said binding maintained activity enzymatic plasmin.

96-well microplates were seeded with 5 mg of purified GapC recombinant protein, washed three times with 0.1% gelatin-PBSA with 0.05% of TWEEN-20 (PBSGT).

The wells were blocked for one hour at 37 ° C in the same solution and washed and incubated with 200 µl of bovine plasmin (0.25 mg / ml, Boehringer Mannheim, Indianapolis, IN) for one hour at 37 ° C. Then, the wells were washed eight times with PBSGT. 200 µL of the synthetic Chromazym-PL substrate (Tos-Gly-Pro-Lys-4-NA, 0.3 mg / ml) was added to the wells and incubated at 37 ° C for one hour. The presence of associated plasmin activity was determined by measuring the amount of paranitroanilide (4-nitraniline) released in the supernatants based on their absorbance at 405 nanometers. A similar procedure was used to measure the plasmin binding activity of intact S. dysgalactiae cells, with the exception that 10 10 cells were washed with PBSGT, resuspended in 400 µl of Chromazym-PL (0 , 3 mg / ml) and incubated for one hour at 37 ° C.

The results, shown in Figure 19, demonstrate that the purified recombinant protein exhibited the ability to bind enzymatically active bovine plasmin. Likewise, when intact S. dysgalactiae cells were used, similar results were obtained. In the figure, the data represent the average of three independent trials.

Thus, the plasmin receptor is located on the surface of S. dysgalactiae and the purified protein maintains its biological activity.

Example 2 Immunization with GapC of S. dysgalactiae and experimental infection in cattle

The vaccines were formulated in such a way that contained 50 mg / ml of recombinant GapC protein purified by affinity in oil-based adjuvant VSA3 (VIDO, Saskatoon, Saskatchewan, Canada). VSA3 is a combination of Emulsigen Plus? (MVP Laboratories, Ralston, Nebraska) and bromide dimethyldioctadecyl ammonium (Kodak, Rochester, New York). In the Figure 18 shows the recombinant GapC protein purified by affinity used for vaccine preparation.

24 non-lactating Holsteins with no history of S. dysgalactiae infection were obtained from different Saskatchewan farms in Canada. One week before vaccination, all animals were treated with Cepha-dry ™ (Ayerst Laboratories, Montreal, Canada) (300 mg per quarter) in order to eliminate any infection of the udder before vaccination.

Groups of eight animals were immunized subcutaneously with two doses of vaccine containing GapC protein from S. dysgalactiae , Mig (a receptor protein for Fc isolated from S. dysgalactiae , which was evaluated simultaneously) or a placebo, with an interval of three weeks between immunizations. Two weeks after the second immunization, the animals were exposed to 650 colony forming units of S. dysgalactiae administered in three of the rooms with an udder infusion cannula. The remaining quarter of each animal was used as a control without infection.

The appearance of signs was examined daily disease clinics in all animals and were collected samples of the udder quarters every day. The sample consistency and cell count was performed somatic as well as the determination of the number of bacteria.

Example 3 Determination of specific antibodies against GapC

The specific antibodies of GapC in bovine serum by a solid phase immunoassay (ELISA). Briefly, microplates were seeded (NUNC, Naperville, Illinois) with 1 µg per well of recombinant antigen purified in 50 mM sodium carbonate buffer, pH 9.6 and incubated overnight at 4 ° C. The liquid was removed and blocked the wells with a solution of bovine serum albumin at 3% for 1 hour at 37 ° C. Then, dilutions were added Serum bovine serum (from 1: 4 to 1: 6400) to the wells and incubated for two hours at room temperature. Later, wells were aspirated, washed and incubated with 100 µl of goat anti-bovine IgG antibodies conjugated with alkaline phosphatase (Kirkgaard & Perry Laboratories Inc., Gaithersburg, MD) for one hour at room temperature. Be the wells were washed again and 100 µl of p-nitrophenol phosphate (SIGMA, St. Louis, MO) as substrate to detect alkaline phosphatase activity. Be determined the absorbance at 405 nanometers after one hour of incubation with the substrate at room temperature.

When referring to the figures, the title of specific antibodies such as the reciprocal of the dilution that showed activity above baseline levels.

Example 4 Bacterial colonization

The number of bacteria spreading was established serial dilutions (from 10 0 to 10 - 3) directly in TSA sheep blood agar plates followed by incubation during overnight at 37 ° C and 5% CO2. Colonization is defined as the recovery of> 500 cfu / ml of the infected organism.

To confirm that the bacteria collected from milk secretions were from S. dysgalactiae , colonies collected from each animal were selected and analyzed using an API strep-20 assay (bioMerieux SA, Hazelwood, Missouri) according to the manufacturer's instructions. This test is a standardized technique that combines 20 biochemical tests to determine the enzymatic activity and the fermentation of sugars. The reactions are read based on a reading table and the identification is obtained both by the index of the analytical profile and by a computer application of identification.

After contact with the microorganism, animals of all groups showed colonization by S. dysgalactiae (Figure 20). Only cows immunized with GapC showed a statistically significant reduction in the number of infected rooms and in the total number of bacteria isolated per quarter. Therefore, immunization with GapC reduced bacterial colonization after contact with S. dysgalactiae .

The relationship between the title of anti-GapC and bacterial colonization is shown in Figures 21 and 22. A large correlation was observed between serum levels of anti-GapC antibodies and the number maximum bacteria (expressed in CFU (log 10) / ml of milk) detected in all rooms (r = 0.74) (Figure 21), as well as with The total number of infected rooms (R2 = 0.74) (Figure 22). The correlation was calculated using the GraphPad Prism program, version 2.01 (GraphPad Software Inc., San Diego, California).

This correlation is also illustrated in the Figures 23 and 24 in which the group immunized with GapC is subdivided  in individuals with a high response and individuals with a low response In those figures, the "title individuals low "refers to the four animals with the worst response in front of GapC, while the "high-title individuals" It refers to the rest of the group. In the high title group you don't know produced colonization while even the low title group showed a reduced number of bacteria collected after three days.

Example 5 Determination of the inflammatory response

The inflammatory response was measured based on the somatic cell count (i.e. lymphocytes, neutrophils and monocytes). Somatic cell counts were carried out in a Coulter counting apparatus using standard techniques, as recommended by Agriculture and Agri-Food Canada Pamphlet IDF50B (1985) "Milk and Milk products-Methods of Sampling" . Samples were always analyzed within the first 48 hours after collection and fixation, on days 1 and 7 after provocation.

The number of somatic cells was determined present in the gland every day after contact with The microorganism In the uninfected room, the count remained constant during the study while on day 1, the group treated with GapC obtained lower results than the group immunized with placebo (Figure 25). The difference between the groups GapC and placebo was statistically significant. The results Day 1 individuals are shown in Figure 26; the results of animals treated with GapC over seven days after coming into contact with the microorganism it shown in Figure 27. Samples of the rooms of the animals immunized with GapC could not be distinguished from the rooms without provocation

Therefore, immunization with GapC reduced the inflammatory response that follows provocation with S. dysgalactiae.

Example 6 Cross protection against S. uberis infection

During the last 3 days of the vaccine trial, a mixed bacterial population recovered from the secretions of the mammary gland. A subsequent analysis with the API 20 Strep test confirmed the identity of the strains present in the mixture as S. dysgalactiae and S. uberis , the second of them representing a natural infection that took place during the test. The group that was immunized with GapC appeared to show significant cross-protection against colonization by S. uberis (see Figure 20), indicating that GapC may be an antigen with a broad cross-protection ability against infection by multiple species of streptococci.

Thus, vaccination with GapC of S. dysgalactiae is therefore capable of providing cross protection against infection by S. uberis .

Thus, the cloning, expression and characterization of various plasmin-binding GapC proteins, as well as methods of using them.

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Bolton, Alexandra J.

Perez-Casal, Joseph

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Claims (13)

1. The use of a GapC protein in the manufacture of the composition of a vaccine useful in the treatment or prevention of mastitis in a mammalian subject, in which the GapC protein is selected from the group consisting of: (a) a GapC protein isolated from Streptococcus dysgalactiae consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 1A-1B (SEQ ID NO: 4) or a amino acid sequence with a sequence identity of at least 80% with the previous one; (b) a GapC protein isolated from Streptococcus agalactiae consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 2A-2B (SEQ ID NO: 6) or a amino acid sequence with a sequence identity of at least 80% with the previous one; (c) a GapC protein isolated from Streptococcus uberis consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 3A-3B (SEQ ID NO: 8) or a amino acid sequence with a sequence identity of at least 80% with the previous one; (d) a GapC protein isolated from Streptococcus parauberis consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 4A-4B (SEQ ID NO: 10) or a amino acid sequence with a sequence identity of at least 80% with the previous one; (e) a GapC protein isolated from Streptococcus iniae consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 5A-5B (SEQ ID NO: 12) or a amino acid sequence with a sequence identity of at least 80% with the previous one. 2. The use of claim 1, wherein the protein is a GapC protein of Streptococcus dysgalactiae consisting of the amino acid sequence shown at the positions of amino acids 1 to 336, inclusive, of Figures 1A-1B (ID SEC No.: 4). 3. The use of claim 1, wherein the protein is a GapC protein of Streptococcus agalactiae consisting of the amino acid sequence shown at the positions of amino acids 1 to 336, inclusive, of Figures 2A-2B (ID of SEC No.: 6). 4. The use of claim 1, wherein the protein is a GapC protein of Streptococcus uberis consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 3A-3B (ID of SEC No.: 8). 5. The use of claim 1, wherein the protein is a GapC protein of Streptococcus parauberis consisting of the amino acid sequence shown at the positions of amino acids 1 to 336, inclusive, of Figures 4A-4B (ID of SEC No.: 10). 6. The use of claim 1, wherein the protein is a GapC protein from Streptococcus iniae consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 5A-5B (ID of SEC No.: 12). 7. The use of a polynucleotide formed by a sequence encoding a GapC protein in the manufacture of the composition of a vaccine useful in the treatment or prevention of mastitis in a mammalian subject, in which the GapC protein is selected from the group consisting of: (a) a GapC protein isolated from Streptococcus dysgalactiae consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 1A-1B (SEQ ID NO: 4) or a amino acid sequence with a sequence identity of at least 80% with the previous one; (b) a GapC protein isolated from Streptococcus agalactiae consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 2A-2B (SEQ ID NO: 6) or a amino acid sequence with a sequence identity of at least 80% with the previous one; (c) a GapC protein isolated from Streptococcus uberis consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 3A-3B (SEQ ID NO: 8) or a amino acid sequence with a sequence identity of at least 80% with the previous one; (d) a GapC protein isolated from Streptococcus parauberis consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 4A-4B (SEQ ID NO: 10) or a amino acid sequence with a sequence identity of at least 80% with the previous one;

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(e) a GapC protein isolated from Streptococcus iniae consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 5A-5B (SEQ ID NO: 12) or a amino acid sequence with a sequence identity of at least 80% with the previous one. 8. The use of claim 7, wherein the protein is a GapC protein of Streptococcus dysgalactiae consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 1A-1B (ID SEC No.: 4). 9. The use of claim 7, wherein the protein is a GapC protein of Streptococcus agalactiae consisting of the amino acid sequence shown at the positions of amino acids 1 to 336, inclusive, of Figures 2A-2B (ID of SEC No.: 6). 10. The use of claim 7, wherein the protein is a GapC protein from Streptococcus uberis consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 3A-3B (ID of SEC No.: 8). 11. The use of claim 7, wherein the protein is a GapC protein from Streptococcus parauberis consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 4A-4B (ID of SEC No.: 10). 12. The use of claim 7, wherein the protein is a GapC protein of Streptococcus iniae consisting of the amino acid sequence shown at amino acid positions 1 to 336, inclusive, of Figures 5A-5B (ID of SEC No.: 12). 13. The use of any of the claims 1 to 12, wherein the mammalian subject is a bovine subject.